WO2021207706A2 - Methods and compositions for inactivating interleukin-2-inducible t-cell kinase (itk) - Google Patents
Methods and compositions for inactivating interleukin-2-inducible t-cell kinase (itk) Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Definitions
- the present disclosure includes methods and compositions for hematopoietic stem cell transplantation treatment such as allogeneic hematopoietic stem cell transplantation (allo-HSCT) that maintain graft-versus-tumor (GVT) effects while reducing or eliminating graft-versus-host disease (GVHD) in e.g., subjects with hematologic malignancies, cancer, or other conditions such as diabetes, or graft-versus- host disease.
- the present disclosure includes suppressors and/of inhibitors of "Inducible T Cell Kinase” (ITK) polypeptides and methods of making and using them, e.g., as agents and pharmaceutical compositions to treat cancer, diabetes, or the like.
- ITK Intelligent T Cell Kinase
- the present disclosure is directed to ITK protein expression and/or activity suppressors or inhibitors.
- the ITK protein inhibitors or suppressors of the present disclosure are used to treat cancers while maintaining graft-versus-tumor (GVT) effects and reducing or eliminating graft-versus-host disease (GVHD).
- GVT graft-versus-tumor
- GVHD graft-versus-host disease
- the present disclosure is directed to ITK protein inhibitors or suppressors as chimeric proteins including fragments or altered or truncated forms of SLP76 protein, or equivalent.
- SLP76 protein or fragments thereof is joined or fused to another moiety (e.g., a targeting domain or dye).
- the disclosure also provides pharmaceutical compositions including the ITK protein inhibitors or suppressors of the present disclosure and methods of making and using them, including methods for ameliorating, treating, suppressing or preventing cancer while maintaining graft-versus-tumor (GVT) effects and/or reducing or eliminating graft-versus-host disease (GVHD).
- the present disclosure also provides compositions for transfecting cells with nucleic acids acting as ITK protein inhibitors or suppressors of the present disclosure and/or the chimeric ITK protein inhibitors or suppressors polypeptides of the present disclosure.
- alloreactive donor T cells are essential for the graft-versus leukemia effect (GVL) (See e.g., Breems, D.A. & Lowenberg, B. Autologous stem cell transplantation in the treatment of adults with acute myeloid leukaemia. Br J Haematol 130, 825-833 (2005); Tugues, S. et al. Graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by GM-CSF-licensed myeloid cells. Sci Transl Med 10 (2018; and Mammadli, M. et al.
- graft-versus- host disease See e.g., Bastien, J.P., Roy, J. & Roy, D.C. Selective T-cell depletion for haplotype-mismatched allogeneic stem cell transplantation. Semin Oncol 39, 674-682 (2012)). Development of GVHD results in significant morbidity and mortality which complicates allo-HSCT, a potentially curative treatment for leukemia.
- Standard immunosuppressive therapy for GVHD is often therapeutically sub-optimal and predisposes patients to opportunistic infections such as Cytomegalovirus (CMV) and relapse of the underlying malignancy.
- CMV Cytomegalovirus
- hematopoietic stem cell transplantation treatment such as allogeneic hematopoietic stem cell transplantation (allo-HSCT) that maintain graft-versus-tumor (GVT) effects while reducing or eliminating graft-versus-host disease (GVHD) in e.g., subjects with hematologic malignancies or cancer.
- allo-HSCT allogeneic hematopoietic stem cell transplantation
- GVT graft-versus-tumor
- GVHD graft-versus-host disease
- the present disclosure relates to method including administering a subject in need thereof an effective amount of an inhibitor of interleukin-2-inducible T-cell kinase (ITK) in accordance with the present disclosure.
- ITK interleukin-2-inducible T-cell kinase
- the present disclosure relates to a composition including an inhibitor of interleukin-2-inducible T-cell kinase (ITK) selected from a small molecule inhibitor, a nucleic acid inhibitor, and a peptide inhibitor or the present disclosure.
- the present disclosure includes one or more nucleic acids encoding peptides of the present disclosure, a vector including a nucleic acid of the present disclosure, or a T-cell including a nucleic acid and/or peptide of the present disclosure.
- a polypeptide of the present disclosure has at least 80% sequence identity to SEQ ID NO: 2, and the polypeptide includes a phosphorylated tyrosine.
- FIGS.1A-1E depicts absence of ITK avoids GVHD while retaining GVL effects during allo-HSCT.
- FIGS.2A-2G depict the regulatory function of ITK in GVHD is T cell-intrinsic.
- FIGS.3A-3J depict innate memory phenotype T cells are not sufficient for GVL effect.
- FIGS.4A-4I depict ITK deficiency results in reduced cytokine production.
- FIGS. 5A-5D depict ITK differentially regulates gene expression in T cells during GVHD.
- FIGS. 6A-6J depict ITK signaling is needed for T cell migration to the GVHD target tissues.
- FIGS.7A-7C depict Itk -/- CD4 + T cells exhibiting attenuated induction of GVHD compared to WT T cells.
- FIGS. 8A-8G depict innate memory phenotype T cells are not sufficient for GVHD effect.
- FIG.9 depicts Itk -/- T cells are capable of cytokine production.
- FIGS. 10A-10D depict quantitative analysis of JAK/STAT and IRF expression and phosphorylation.
- FIGS.11A and 11B depict quantitative analysis of tissue BLI.
- FIG.12. depicts a general strategy of the inventions of the present disclosure.
- FIGS. 13A-13E depict disruption of ITK:SLP76 Y145 signaling allows tumor clearance without inducing GVHD.
- FIGS. 14A-14H depict SLP76Y145FKI donor CD8 + and CD4 + T cells exhibit reduced cytokine production and reduced proliferation.
- FIGS.15A-15H depict Eomes is required for cytotoxicity and GVL effect by both WT and SLP76Y145FKI T cells.
- FIGS.16A-16G depict SLP76Y145/ITK signaling is required for T cell migration to the GVHD target tissues.
- FIGS. 17A-17F depict development of a novel peptide that disrupts the interaction between SLP76 and ITK.
- FIGS. 20A-20C depict SLP76 Y145FKI CD4 + T cells exhibit attenuated induction of GVHD compared to WT T cells.
- FIGS.21A-21F depict the innate memory phenotype of CD8 + T cells does not separate GVHD and GVL effects.
- FIG. 22 depicts SLP76pTYR peptide and the related fusion sequences and plasmid.
- FIGS.23 depicts SLP76Y145FKI T cells are capable of cytokine production.
- FIGS.24A and 24B depict Eomes deletion on CD8 + and CD4 + T cells.
- FIGS.25A and 25B depict quantitative analysis of donor T cells in secondary lymphoid organs and GVHD target organs.
- FIGS. 26A and 26B depict quantitative analysis of tissue bioluminescence imaging (BLI).
- FIGS.27A-27F depict ITK inhibitors 10n and CTA056 are not specific for ITK.
- FIGS. 28A-28H depict quantitative analysis of SLP76:ITK signaling protein expression in cells treated with peptide SLP76pTYR.
- FIGS. 28A-28H depict quantitative analysis of SLP76:ITK signaling protein expression in cells treated with peptide SLP76pTYR.
- the present disclosure relates to compositions and methods including administering to a subject in need thereof an effective amount of an inhibitor of interleukin-2-inducible T-cell kinase (ITK).
- ITK interleukin-2-inducible T-cell kinase
- the inventors show herein that targeting ITK signaling can inhibit GVHD while maintaining GVL function allogeneic transplant model. This finding will have a significant impact on the future development of transplantation strategies. Further, the inventors have developed a specific inhibitor that disrupts SLP76:ITK interactions, leading to reduced inflammatory cytokine production and reduced chemokine receptor upregulation. Moreover, T cells treated with SLP76pTYR exhibit increased numbers of CD25-, FoxP3+ regulatory T cells.
- kinase domain inhibitors disrupt all the activity- dependent pathways, even those that are not involved in driving the malignancy but are required for other critical pathways.
- the present disclosure provides innovative methodology or strategy focusing on specifically disrupting the kinase docking to a specific scaffold-of-interest without inhibiting its activity or affecting its ability to be involved in other pathways. This approach is critical for eliciting its desirable GVL effects during allo-HSCT. This new and different approach to inhibiting ITK signaling is expected to allow for overcoming the limitations of current ITK inhibitors. [0046] The inventors have also shown that SLP76145pTYR specifically inhibits the phosphorylation of ITK and downstream signaling molecules, including PLC- ⁇ 1, and ERK, without affecting the phosphorylation of other signaling molecules.
- T cells from GVHD patient blood samples with SLP76pTYR enhances the development of FoxP3 + regulatory T cells, while significantly reducing IFN- ⁇ and TNF- ⁇ production by T cells from GVHD patient blood samples.
- ITK interleukin- 2-inducible T-cell kinase
- a subject such as a human or non-human mammal (such as a dog, cat, monkey, horse, mouse, rat, or the like) is suffering from a disorder with an inflammatory component.
- the subject is suffering from a cancer and is receiving a T-cell-based immunotherapy.
- the subject is suffering from a T-cell lymphoma.
- the subject is suffering from diabetes.
- the subject is suffering from atherosclerosis.
- the subject is suffering from GVHD.
- the subject has had or is planning to have organ transplantation.
- the subject has had or is planning to have a skin graft.
- the methods of this disclosure can prevent or treat GVHD.
- Methods of Treatment [0049]
- the present disclosure is directed to methods of treating a subject by administering the subject an effective amount of an inhibitor of interleukin- 2-inducible T-cell kinase (ITK) of the present disclosure.
- ITK interleukin- 2-inducible T-cell kinase
- the term "effective amount" means the total amount of each active component of a pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention of the relevant medical condition, amelioration of the symptoms, or an increase in rate of treatment, healing, prevention or amelioration of such conditions, or inhibition of the progression of the condition.
- the term refers to that ingredient alone.
- the term refers to combined amounts of the active ingredients that result in a desired therapeutic effect, whether administered in combination, serially or simultaneously.
- the disclosure is directed to methods involving immunotherapy approaches.
- the methods of the instant disclosure include providing a T cell to a subject, wherein the interleukin-2-inducible T-cell kinase (ITK) gene in the T cell has been inactivated.
- the providing includes obtaining T cells, inactivating the ITK gene in the obtained cells ex vivo, and transplanting the cells into the subject.
- the T cell is autologous.
- the T cell is non-autologous.
- the inactivation of the ITK gene in the T cell is achieved by deleting or mutating the ITK gene in whole or in part such that no functional ITK protein product is expressed. In some embodiments, the inactivation of the ITK gene in the T cell is achieved by a method selected from the group consisting of CRISPR/Cas system, Cre/Lox system, TALEN system and homologous recombination. [0052] In some embodiments, the inactivation of the ITK genes in the T cell is achieved by blocking the signaling of ITK using an ITK inhibitor. In some embodiments, the ITK inhibitor is selected from the group consisting of a small molecule inhibitor, a nucleic acid inhibitor, and a peptide inhibitor.
- the ITK inhibitor is a nucleic acid inhibitor selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.
- the ITK inhibitor is a peptide inhibitor including a sequence as shown in SEQ ID NO: 2, wherein the peptide inhibitor includes a phosphorylated tyrosine.
- the peptide inhibitor further comprises a TAT-peptide sequence such as (SEQ ID NO: 3).
- the peptide inhibitor further comprises tag.
- the tag is a fluorescent tag.
- compositions to inhibit the ITK protein or ITK signaling.
- the compositions of the disclosure include an ITK inhibitor.
- the ITK inhibitor is selected from the group consisting of a small molecule inhibitor, a nucleic acid inhibitor, and a peptide inhibitor.
- the ITK inhibitor is a nucleic acid inhibitor selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.
- the ITK inhibitor is a peptide inhibitor that includes a sequence as shown in SEQ ID NO: 2, and wherein the peptide inhibitor includes a phosphorylated tyrosine.
- the ITK inhibitor is a peptide inhibitor that includes an amino acid sequence having at least 80%, 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 2, and wherein the peptide inhibitor includes a phosphorylated tyrosine.
- the peptide inhibitor further includes a TAT-peptide sequence (SEQ ID NO: 3).
- the peptide inhibitor further includes tag.
- the tag is a fluorescent tag.
- T-cell kinase interleukin-2- inducible T-cell kinase
- TCR T cell receptor
- ITK inducible T cell kinase
- ITK is involved in signaling which leads to cytokine production by T cell populations, and also regulates the development of a distinct, innate-type cytokine-producing T cell population in the thymus (See e.g., Atherly, L.O. et al. The Tec family tyrosine kinases Itk and Rlk regulate the development of conventional CD8+ T cells. Immunity 25, 79-91 (2006)), referred to as innate memory phenotype (IMP) T cells. These cells express significantly higher levels of CD122, CD44, and Eomes.
- IMP innate memory phenotype
- IMP T cells derived from WT-bone marrow exhibit GVL effects but also cause GVHD, while IMP T cells derived from Itk -/- bone marrow cells are able to clear the leukemia cells without inducing GVHD.
- T cells from IL-4 receptor-alpha and ITK-double knockout mice which lack the IMP phenotype, did not induce GVHD, indicating that absence of ITK, and not IMP cells, is responsible for reduced GVHD in the absence of ITK.
- Both CD8 + and CD4 + T cells donor T cells from Itk -/- mice also exhibit increased expression of perforin and significantly reduced expression of pro-inflammatory cytokines. Itk -/- donor T cells also exhibit reduced proliferation, which was cell-extrinsic.
- CD4 + and CD8 + T cells from ITK-deficient mice show defects in T cell migration into GVHD target tissues, caused by reduced expression of chemokine receptors. This leads to decreased tissue damage during allo-HSCT. Itk -/- T cells can successfully clear leukemia cells in circulation, however they are unable to clear subcutaneously growing leukemic cells due to this migration defect.
- RNA sequencing data revealed that ITK deficiency impacts genes involved in cytokine production, cell adhesion, and chemokine and cytokine receptor expression. These genes are involved in the pathogenesis of GVHD. The studies identified a specific and novel potential therapeutic target and its downstream mechanism for separating GVHD and GVL after allo-HSCT.
- cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks introns or intron sequences that may be present in corresponding genomic DNA. In embodiments, cDNA may refer to a nucleotide sequence that correspond to the nucleotide sequence of an mRNA from which it is derived.
- Coding sequence The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
- fragment means a polypeptide having one or more amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain. In embodiments, a fragment contains at least 1% to 75%, at least 2% to 40% or about 2 to 30% of the number of amino acids of the mature polypeptide of SEQ ID NO: 4.
- mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications.
- polynucleotide and nucleic acid refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
- terms “polynucleotide” and “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
- polynucleotide and “nucleic acid” should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
- complementary refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands or regions. Complementary polynucleotide strands or regions can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of stable duplexes.
- Complementarity is typically measured with respect to a duplex region and thus excludes, for example, overhangs.
- a duplex region may include a region of complementarity between two strands or between two regions of a single strand, for example, a unimolecular siRNA.
- the region of complementarity results from Watson-Crick base pairing.
- perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand or region can hydrogen bond with each nucleotide unit of a second polynucleotide strand or region.
- nucleotide units of two strands or two regions can hydrogen bond with each other.
- the polynucleotide strands or regions exhibit 10% complementarity.
- the polynucleotide strands exhibit 90% complementarity.
- Substantial complementarity refers to polynucleotide strands or regions exhibiting 80% or greater complementarity.
- RNA complementary DNA
- anneal or “hybridize”
- a nucleic acid specifically binds to a complementary nucleic acid
- Standard Watson-Crick base-pairing includes: adenine/adenosine) (A) pairing with thymidine/thymidine (T), A pairing with uracil/uridine (U), and guanine/guanosine) (G) pairing with cytosine/cytidine (C).
- A adenine/adenosine
- T thymidine/thymidine
- U uracil/uridine
- G guanine/guanosine
- C cytosine/cytidine
- G cytosine/cytidine
- G can also base pair with U.
- G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA.
- a G e.g., of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA) base pairing with a guide RNA
- a G e.g., of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA) base pairing with a guide RNA
- a target nucleic acid e.g., target DNA
- Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible.
- the conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences.
- the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
- sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
- a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ⁇ bulge ⁇ , and the like).
- a polynucleotide can include 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize.
- an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
- the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.
- Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method.
- Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol.
- peptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
- a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine.
- Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine- glutamine.
- degree of identity refers to the relatedness between two amino acid sequences or between two nucleotide sequences and is described by the parameter "identity”.
- the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) aligning the two sequences by any suitable alignment program using the default scoring matrix and default gap penalty; 2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical amino acid or nucleotide in the two aligned sequences on a given position in the alignment; and 3) dividing the number of exact matches with the length of the reference sequence.
- the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) aligning the two sequences by any suitable alignment program using the default scoring matrix and default gap penalty; 2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical amino acid; or nucleotide in the two aligned sequences on a given position in the alignment; and 3) dividing the number of exact matches with the length of the longest of the two sequences.
- the degree of sequence identity refers to and may be calculated as described under “Degree of Identity” in U.S. Patent No.10,531,672 starting at Column 11, line 56. U.S. Patent No.10,531,672 is incorporated by reference in its entirety.
- an alignment program suitable for calculating percent identity performs a global alignment program, which optimizes the alignment over the full- length of the sequences.
- the global alignment program is based on the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970), "A general method applicable to the search for similarities in the amino acid sequence of two proteins", Journal of Molecular Biology 48 (3): 443-53). Examples of current programs performing global alignments using the Needleman-Wunsch algorithm are EMBOSS Needle and EMBOSS Stretcher programs, which are both available on the world wide web at www.ebi.ac.uk/Tools/psa/.
- a global alignment program uses the Needleman-Wunsch algorithm and the sequence identity is calculated by identifying the number of exact matches identified by the program divided by the "alignment length", where the alignment length is the length of the entire alignment including gaps and overhanging parts of the sequences.
- the term "deoxynucleotide” refers to a nucleotide or polynucleotide lacking an OH group at the 2' or 3' position of a sugar moiety, and/or a 2',3' terminal dideoxy, but instead having a hydrogen at the 2' and/or 3' carbon.
- deoxyribonucleotide and “DNA” refer to a nucleotide or polynucleotide including at least one ribosyl moiety that has an H at the 2' position of a ribosyl moiety.
- a deoxyribonucleotide is a nucleotide having an H at its 2' position.
- isolated means a substance in a form or environment that does not occur in nature.
- nucleotide refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof.
- Nucleotides include species that include purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs.
- a "nucleotide” includes a cytosine, uracil, thymine, adenine, or guanine moiety.
- nucleotides include unmodified cytosine, uracil, thymine, adenine, and guanine.
- nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety as defined herein.
- isolated nucleic acid fragment and “isolated nucleic acid molecule” are used interchangeably and are optionally single-stranded or double- stranded with synthetic, non-natural or modified nucleotide bases. This will indicate a single-stranded RNA or DNA polymer.
- nucleic acid molecule refers to any molecule containing multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)).
- bases include C, T, U, C, and G, as well as variants thereof.
- the term refers to ribonucleotides (including oligoribonucleotides (ORN)) as well as deoxyribonucleotides (including oligodeoxynucleotides (ODN)).
- the term shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base containing polymer.
- Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but include synthetic (e.g., produced by oligonucleotide synthesis).
- nucleic acid “nucleic acid molecule” and “polynucleotide” may be used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs.
- Polynucleotides can have any three- dimensional structure.
- a nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand).
- Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA) and portions thereof, transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
- mRNA messenger RNA
- RNA messenger RNA
- transfer RNA transfer RNA
- ribosomal RNA siRNA
- micro-RNA micro-RNA
- ribozymes cDNA
- recombinant polynucleotides branched polynucleotides
- plasmids vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
- a component of interest may be “substantially purified” when the preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components.
- a "substantially purified" component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater.
- a "vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an "insert", may be attached so as to bring about the replication of the attached segment in a cell.
- An "expression cassette” includes a DNA coding sequence operably linked to a promoter. "Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
- recombinant expression vector or "DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and one insert.
- Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences.
- the insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.
- Any given component, or combination of components can be unlabeled, or can be detectably labeled with a label moiety. In some cases, when two or more components are labeled, they can be labeled with label moieties that are distinguishable from one another.
- the present disclosure includes compositions and methods for the treatment, amelioration, prevention or suppression of cancers, malignancies, blood malignancies, and/or immunological deficiencies for subjects in need thereof.
- the present disclosure provides methods and compositions for the treatment, amelioration, prevention or suppression of disease such as where a subject suffers from cancer, diabetes, or graft-versus- host disease.
- the present disclosure includes one or more inhibitors of "Inducible T Cell Kinase” (ITK) polypeptides and methods of making and using them, e.g., as agents and pharmaceutical compositions to treat cancer, diabetes, or graft- versus- host disease, or other pathologic responses.
- ITK Inducible T Cell Kinase
- the present disclosure is directed to ITK protein expression and/or activity inhibitors.
- the ITK protein inhibitors of the disclosure are used to treat cancer, diabetes, or graft- versus- host disease, or other pathologic responses.
- the present disclosure is directed to ITK protein inhibitors as chimeric proteins including fragments or altered or truncated forms of mammalian or human SLP76 protein (SEQ ID NO:4) or equivalent.
- SLP76 protein, or fragments thereof are joined or fused to another moiety (e.g., a dye or TAT sequence).
- the present disclosure also provides pharmaceutical compositions including the ITK protein inhibitors of the disclosure, and methods of making and using them, including methods for ameliorating or preventing cancer, diabetes, or graft-versus- host disease or other pathologic responses.
- the disclosure also provides compositions for transfecting cells with nucleic acids acting as ITK protein inhibitors and/or the chimeric ITK protein inhibitors polypeptides of the present disclosure. [0095]
- the present disclosure includes methods and compositions for transfecting nucleic acids into a cell including an ITK protein inhibitor of the present disclosure.
- the ITK protein inhibitor of the present disclosure includes a nucleic acid including naked DNA or RNA, and optionally the naked DNA or RNA, or RNAi such as siRNA or miRNA, is operably linked to a promoter.
- the nucleic acid includes plasmid DNA, a recombinant virus or phage, an expression cassette or a vector such as an expression vector.
- the cell is a mammalian cell, wherein optionally the mammalian cell is a human cell such as a human T cell, or alloreactive donor T cells suitable for use in allogeneic hematopoietic stem cell transplantation (allo-HSCT).
- the present disclosure includes methods for transfecting a cell with nucleic acid of the present disclosure including the following steps: (a) providing a nucleic acid-including composition of the present disclosure (for transfecting nucleic acids); (b) contacting the cell with the composition of step (a) under conditions wherein the composition is internalized into the cell.
- the transfecting is an in vivo transfection or an in vitro transfection.
- the present disclosure includes methods for preventing, inhibiting, suppressing or ameliorating cancer, diabetes, or graft-versus- host disease or other pathologic responses by inhibiting the activation of T-helper cells responsible for the graft-versus- host disease while maintaining a graft-versus tumor effect, the method including, a peptide conjugate that is able to efficiently gain entry into T-helper cell and inhibit the activation of ITK and SLP-76, leading to inhibition of T-helper cell responsible graft-versus- host disease.
- the cell is a T cell or any T cell suitable for use in hematopoietic stem cell transplantation such as allogeneic hematopoietic stem cell transplantation (allo-HSCT).
- a suitable peptide of the present disclosure includes the peptide of SEQ ID NO 2.
- a suitable peptide such as a peptide inhibitor of the present disclosure includes the peptide having an amino acid sequence having at least 80%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO 2, wherein the peptide inhibitor includes a phosphorylated tyrosine.
- the present disclosure provides isolated, synthetic or recombinant polypeptides or peptides including or consisting of SEQ ID NO: 2, wherein the peptide inhibitor includes a phosphorylated tyrosine.
- a suitable peptide of the present disclosure disrupts SLP76:ITK interactions, leading to reduced inflammatory cytokine production and reduced chemokine receptor upregulation.
- the present disclosure relates to a complementary deoxynucleotide (cDNA) sequence encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2.
- the present disclosure relates to a complementary deoxynucleotide (cDNA) sequence encoding the amino acid sequence of SEQ ID NO: 2.
- the cDNA are made through laboratory manipulation and genetic engineering techniques. In embodiments, methods of making synthetic polypeptides of the present disclosure are known in the art.
- a suitable cDNA sequence of the present disclosure includes the nucleic acid sequence of SEQ ID NO: 1.
- SEQ ID NO: 1 encodes SEQ ID NO: 2.
- the nucleic acid sequence of SEQ ID NO: 1 may include conservative substitutions, wherein alterations such as substitutions may be present and the nucleotide encodes SEQ ID NO: 2.
- the polypeptides and peptides of the present disclosure have increased intracellular penetration and delivery because of several added moieties.
- the polypeptides and peptides of the present disclosure may include a peptide conjugate including a subsequence of SLP76 that binds to ITK.
- the polypeptides and peptides of the present disclosure are able to efficiently penetrate and enter T cells such as CD4 positive T cells.
- Pharmaceutical Compositions including an ITK-inhibitory nucleic acid or peptide or polypeptide of the present disclosure and a pharmaceutically acceptable excipient.
- the present disclosure provides for uses of an ITK-inhibitory nucleic acid or peptide or polypeptide of the disclosure to make a pharmaceutical composition.
- the present disclosure provides parenteral formulations including an ITK-inhibitory nucleic acid or polypeptide of the present disclosure.
- the present disclosure provides enteral formulations including an ITK-inhibitory nucleic acid or polypeptide of the present disclosure.
- the present disclosure provides methods for treating GVHD including providing a pharmaceutical composition including an ITK-inhibitory nucleic acid or polypeptide of the present disclosure; and administering an effective amount of the pharmaceutical composition to a subject in need thereof.
- the pharmaceutical compositions used in the methods of the present disclosure can be administered by any means known in the art, e.g., parenterally, topically, orally, or by local administration, such as by aerosol or transdermally.
- the pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like.
- compositions of the present disclosure can be prepared according to any method known to the art for the manufacture of pharmaceuticals.
- Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
- a formulation of the invention can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
- Formulations of the disclosure may include one or more diluents, emulsifiers, preservatives, buffers, excipients, etc.
- a vaporized medicine can be inhaled through a tube-like mouthpiece, e.g., an inhaler, nebulizer or atomizer; this can have a benefit of allowing surrounding air to mix with the formulation, decreasing the unpleasantness of the vapor, if any.
- compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
- Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
- Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen.
- Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
- formulations can be given depending on the dosage and frequency as required and tolerated by the patient.
- the formulations should provide a sufficient quantity of active agent to effectively treat the treat (e.g., ameliorate) or prevent disease and/or its symptoms of GVHD.
- the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol.58:611- 617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm.
- the present disclosure relates to a a method including administering a subject in need thereof an effective amount of an inhibitor of interleukin-2-inducible T-cell kinase (ITK).
- ITK interleukin-2-inducible T-cell kinase
- the subject suffers from cancer, diabetes, or graft-versus- host disease.
- the ITK inhibitor is selected from the group consisting of a small molecule inhibitor, a nucleic acid inhibitor, and a peptide inhibitor.
- the nucleic acid inhibitor is selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.
- the peptide inhibitor includes a sequence as shown in SEQ ID NO: 2, and wherein the peptide inhibitor includes a phosphorylated tyrosine.
- the peptide inhibitor further includes a TAT-peptide sequence.
- the ITK inhibitor is administered intermittently. In embodiments, the ITK inhibitor is administered every other day, every three days, every five days or once a week. In embodiments, the ITK inhibitor is administered every hour, every two hours, every three hours, every six hours or every twelve hours. In embodiments, the ITK inhibitor is administered by intravenous (i.v.) injection, intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, or aerosolized delivery. In embodiments, the effective amount of the ITK inhibitor is between 0.2 mg/kg and 20 mg/kg.
- the present disclosure relates to a method including providing a T cell to a subject, wherein the interleukin-2-inducible T-cell kinase (ITK) gene in the T cell has been inactivated.
- providing includes obtaining T cells, inactivating the ITK gene in the obtained cells ex vivo, and transplanting the cells into the subject.
- providing includes inactivating the ITK gene in insulin-producing cells in the subject in vivo.
- the inactivation of the ITK gene in the T cell is achieved by deleting or mutating the ITK gene in whole or in part such that no functional ITK protein product is expressed.
- the inactivation of the ITK gene in the T cell is achieved by a method selected from the group consisting of CRISPR/Cas system, Cre/Lox system, TALEN system and homologous recombination. In some embodiments, the inactivation of the ITK genes in the T cell is achieved by blocking the signaling of ITK using an ITK inhibitor.
- the ITK inhibitor is selected from a small molecule inhibitor, a nucleic acid inhibitor, and a peptide inhibitor. In some embodiments, the nucleic acid inhibitor is selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.
- the peptide inhibitor comprises a sequence as shown in SEQ ID NO: 2, and wherein the peptide inhibitor comprises a phosphorylated tyrosine. In some embodiments, the peptide inhibitor comprises an amino acid sequence having at least 80%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 2 and wherein the peptide inhibitor includes a phosphorylated tyrosine. In embodiments, the peptide inhibitor further includes a TAT- peptide sequence. [00108] In embodiments, the present disclosure includes a composition including an inhibitor of interleukin-2-inducible T-cell kinase (ITK) selected from a small molecule inhibitor, a nucleic acid inhibitor, and a peptide inhibitor.
- ITK interleukin-2-inducible T-cell kinase
- the nucleic acid inhibitor is selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.
- the peptide inhibitor includes a sequence as shown in SEQ ID NO: 2, and wherein the peptide inhibitor includes a phosphorylated tyrosine.
- the peptide inhibitor further includes a TAT- peptide sequence.
- the present disclosure includes a nucleic acid encoding a peptide inhibitor of the present disclosure.
- the present disclosure includes a vector including a nucleic acid according to the present disclosure.
- the present disclosure relates to one or more T-cells including a nucleic acid encoding a polypeptide of the present disclosure, or the T- cells include a polypeptide having at least 90%, at least 95%, at least 99% sequence identity to SEQ ID NO: 2, wherein the peptide inhibitor includes a phosphorylated tyrosine.
- EXAMPLE I Materials and Methods [00109] Mice: Itk -/- mice were described previously (See e.g., Liao XC, Littman DR. Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk. Immunity (1995) 3:757–69. doi: 10.1016).1074-7613(95)90065-9).
- mice C57BL/6, C57BL/6.SJL (B6-SJL), ROSA26-pCAGGs-LSL-Luciferase, Thy1.1 (B6.PL- Thy1a/CyJ), CD45.1 (B6.SJL-Ptprc a Pepc b /BoyJ) and BALB/c mice were purchased from Charles River or Jackson Laboratory. Eomes flox/flox mice, B6.129S1 mice, and CD4cre mice were purchased from Jackson Laboratory. Mice expressing Cre driven by the CMV promoter (CMV-Cre) were purchased from the Jackson Laboratory and crossed to ROSA26-pCAGGs-LSL-Luciferase mice (B6-luc).
- CMV-Cre CMV promoter
- mice were bred with Itk -/- mice to create Itk -/- luc mice.
- Itk -/- and Il4ra -/- double knockout mice have been described (See e.g., Huang W, et al., ITK tunes IL-4-induced development of innate memory CD8+ T cells in a gammadelta T and invariant NKT cell-independent manner. J Leukoc Biol (2014) 96:55–63. doi: 10.1189/jlb.1AB0913-484RR). [00110] Mice aged 8-12 weeks were used, and all experiments were performed with age and sex-matched mice.
- T cells were purified with either anti-CD8 or anti-CD4 magnetic beads using MACS columns (Miltenyi Biotec, Auburn, CA) prior to cell surface staining.
- FACS sorting was performed with a FACS Aria III cell sorter (BD Biosciences). FACS-sorted populations were typically of > 95% purity. Antibodies against IRF4, STAT3, JAK2, JAK1, GAPDH, and ⁇ -Actin (for total and/or phospho proteins) were purchased from Cell Signaling Technology (Danvers, MA). All cell culture reagents and chemicals were purchased from Invitrogen (Grand Island, NY) and Sigma-Aldrich (St. Louis, MO), unless otherwise specified. The A20 cell lines (American Type Culture Collection; Manassas, VA), and primary mouse B-ALL blast cells (See e.g., Cheng Y, et al.
- mice Lethally irradiated BALB/c mice (800 cGy) were injected intravenously with 10 ⁇ 10 6 T cell-depleted bone marrow (TCDBM) cells with or without 1 ⁇ 10 6 FACS-sorted CD8 + T cells, 1 ⁇ 10 6 CD4 + T cells, or CD8/CD4 cells mixed at a 1:1 ratio. FACS-sorted total CD8 + , total CD4 + , or mixed donor CD4 + and CD8 + T cells from WT (C57Bl/6) or Itk -/- mice were used.
- B-cell acute lymphoblastic leukemia (See e.g., Cheng Y, et al. LNK/SH2B3 regulates IL-7 receptor signaling in normal and malignant B-progenitors. J Clin Invest (2016) 126:1267–81. doi: 10.1172/JCI81468) transduced with luciferase were cultured as described previously (Edinger M, et al. CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med (2003) 9:1144–50.
- mice were evaluated twice a week from the time of leukemia cell injection for 65 days by bioluminescence imaging using the IVIS 50 Imaging System (Xenogen) as previously described (See e.g, Contag CH, et al., Advances in in vivo bioluminescence imaging of gene expression. Annu Rev Biomed Eng (2002) 4:235–60. doi: 10.1146/ annurev.bioeng.4.111901.093336).
- mice Clinical presentation of the mice was assessed 2- 3 times per week by a scoring system that sums changes in 5 clinical parameters: weight loss, posture, activity, fur texture, and skin integrity (See e.g., Cooke KR, et al. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin. Blood (1996) 88:3230– 9. doi: 10.1182/blood.V88.8.3230.bloodjournal8883230). Mice were euthanized when they lost ⁇ 30% of their initial body weight or became moribund.
- Itk -/- (CD45.1 + ) and WT (CD45.2) T cells were FACS-sorted from Thy1.1 hosts and then transplanted to irradiated BALB/c mice carrying leukemia cells, along with T cell-depleted bone marrow as described above. This was followed by analysis of GVHD and GVL.
- FACS-sorted CD8 + T cells from WT or Itk -/- mice were mixed at a 1:1 ratio and injected into BALB/c mice (2 ⁇ 10 6 CD8 + T cells total).
- Tissues Imaging Allo-HSCT was performed with 10 ⁇ 10 6 WT T cell- depleted BM cells and 1 ⁇ 10 6 FACS-sorted CD8 + or 1 ⁇ 10 6 FACS-sorted CD4 + T cells (from B6-luc or Itk -/- luc mice) and bioluminescence imaging of tissues was performed as previously described (See e.g., Beilhack A, et al. In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets. Blood (2005) 106:1113–22. doi: 10.1182/blood-2005-02-0509). Briefly, 5 minutes after injection with luciferin (10 ⁇ g/g body weight), selected tissues were prepared and imaged for 5 minutes.
- Cytotoxicity assays For cytotoxicity assays, luciferase-expressing A20 cells were seeded in 96-well flat bottom plates at a concentration of 3x10 5 cells/ml.
- D-firefly luciferin potassium salt 75 ⁇ g/ml; Caliper Hopkinton, MA
- luciferin potassium salt 75 ⁇ g/ml; Caliper Hopkinton, MA
- ex vivo effector cells (MACS-sorted or FACS-sorted CD8 + T cells from bone marrow-transplanted mice) were added at 40:1, 10:1, and 5:1 effector-to-target (E:T) ratios and incubated at 37°C for 4 hours.
- Bioluminescence in relative luciferase units (RLU) was then measured for 1 minute. Cells treated with 1% Nonidet P-40 was used as a measure of maximal killing. Target cells incubated without effector cells were used to measure spontaneous death.
- % specific lysis 100 ⁇ (spontaneous death RLU–test RLU)/(spontaneous death RLU– maximal killing RLU) (See e.g., Karimi MA, et al. Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay. PloS One (2014) 9:e89357. doi: 10.1371/journal.pone.0089357).
- the small intestines were washed in media, shaken in strip buffer at 37°C for 30 minutes to remove the epithelial cells, and then washed, before digesting with collagenase D (100 mg/ml) and DNase (1mg/ml) for 30 minutes in 37°C, and followed by filtering with a 70 ⁇ m filter. Lymphocytes from the liver and intestines were further enriched using a 40% Percoll gradient. The cells were analyzed for H2K b , CD45.1 + and CD45.2 + CD8 + T cells by flow cytometry, but any bone marrow-derived T cells (Thy1.1 + ) were excluded.
- RNA sequencing T cells from WT C57Bl/6 or Itk -/- mice were MACS purified and FACS sorted, and 2 ⁇ 10 6 FACS sorted CD8 + T cells were transplanted into BALB/c mice, along with TCD BM as described above. Seven days post transplantation, donor cells were purified from spleen (Spl). Samples were submitted to SUNY Upstate Medical University Sequencing core facility for RNA sequencing. Sort enough donor T cells from small intestine of the recipient mice that received Itk -/- T cells was problematic.
- RNA sequencing data was generated from five groups: WT- Pre and Itk -/- -Pre cells prior to transplantation; WT-Spleen, and Itk -/ - Spleen using cells isolated from 7 days post-transplantation. Copy numbers were further analyzed in Gene Spring for normalization, quality control, correlation, principal component analysis and gene differential expression. The sequencing data is deposited in www.ncbi.nml.nih.gov/geo/ Accession Display GSE161160. [00121] Western blotting: Cells were lysed in freshly prepared lysis buffer using RIPA buffer from Fisher Scientific (cat#PI89900) and Complete Protease Inhibitor Cocktail (cat#11697498001) and centrifuged for 10 minutes at 4°C.
- qPCR assay To confirm the differences observed in RNA sequencing, pre- and post-transplanted donor T cells were FACS sorted from recipient mice on H2K b markers, and total RNA was isolated from T cells using the RNeasy kit from Qiagen (Germantown, MD). cDNA was made from total RNA using a cDNA synthesis kit (Invitrogen). qRT-PCR assay was performed with a premade customized plate (Fisher Scientific, Hampton, NH).
- Statistics. All numerical data are reported as means with standard deviation. Data were analyzed for significance with GraphPad Prism. Differences were determined using one-way or two-way ANOVA and Tukey’s multiple comparisons tests, or with a student’s t-test when necessary. P-values less than or equal to 0.05 are considered significant. All transplant experiments are done with N 5 mice per group, and repeated at least twice, according to power analyses. Mice are sex- matched, and age-matched as closely as possible.
- T cell-depleted bone marrow cells from B6.PL-Thy1 a /CyJ (Thy1.1) mice, and T cells from C57BL/6 (B6) WT or Itk -/- mice.
- mice Lethally irradiated BALB/c mice were injected intravenously with 10 ⁇ 10 6 wild-type (WT) T cell-depleted donor BM cells along with 2 ⁇ 10 6 FACS-sorted donor T cells (1 ⁇ 10 6 CD8 + and 1 ⁇ 10 6 CD4 + ), followed by intravenous challenge with 2 ⁇ 10 5 luciferase-expressing B-ALL-luc blast cells as described (See e.g., Liao XC, Littman DR. Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk. Immunity (1995) 3:757–69. doi: 10.1016/1074-7613(95)90065-9).
- mice were monitored for cancer cell growth using IVIS bioluminescence imaging for over 60 days (FIG.1A). While leukemia cell growth was observed in T cell-depleted BM-transplanted mice without T cells, leukemia cell growth was not seen in mice transplanted with T cells from either WT or Itk -/- mice. As expected, mice transplanted with WT T cells cleared the leukemia cells but suffered from GVHD. In contrast, mice transplanted with Itk -/- T cells cleared the leukemia cells and displayed minimal signs of GVHD. Most animals transplanted with Itk -/- T cells survived for more than 65 days post-allo-HSCT (FIG.
- Donor CD8 + T cells are more potent than CD4 + T cells in mediating GVL effects, but both CD4 + and CD8 + T cells mediate severe GVHD in mice and humans (See e.g., Amir AL, et al. Identification of a coordinated CD8 and CD4 T cell response directed against mismatched HLA Class I causing severe acute graft- versus-host disease. Biol Blood Marrow Transplant (2012) 18:210–9. doi: 10.1016/j.bbmt.2011.10.018 24; Yu XZ, et al. Alloantigen affinity and CD4 help determine severity of graft-versus-host disease mediated by CD8 donor T cells. J Immunol (2006) 176:3383–90.
- FIGS.1A-1E depict the absence of ITK avoids GVHD while retaining GVL effects during allo-HSCT.
- Group 2 received 10X10 6 T cell depleted bone marrow along with 2X10 5 B-ALL-luc cells (TCDBM+B-ALL luc +), Group 3 was transplanted with 1X10 6 purified WT CD8 + and 1X10 6 CD4 + T cells (1:1 ratio) along with 2X10 5 B-ALL-luc+ cells 10X10 6 (TCDBM+B-ALL luc + WT CD8+CD4).
- Group 4 received 1X10 6 purified Itk -/- CD8 + and 1X10 6 CD4 + T cells (1:1 ratio) along with 2X10 5 B-ALL-luc+ cells 10X10 6 (TCDBM+B-ALL luc + Itk -/- CD8+CD4).
- FIG.1A depicts recipient BALB/c mice were imaged using IVIS 3 times a week.
- FIG.1B depicts that the mice were monitored for survival
- FIG. 1C depicts changes in body weight
- FIG. 1D depicts clinical score for 65 days post BMT.
- FIG. 1E depicts quantified luciferase bioluminescence of leukemia cell growth.
- FIG. 7A the figure depicts Itk -/- CD4 + T cells exhibit attenuated induction of GVHD compared to WT T cells.
- FIG.7A depicts 10X10 6 T cell- depleted bone marrow and 1X10 6 purified CD4 + T cells from WT or Itk -/- CD4 + T cells were transplanted into irradiated BALB/c mice. The mice were monitored for survival, FIG.7B depicts changes in body weight, and FIG.7C depicts clinical score for 70 days post-BMT.
- T Cells Innate memory phenotype (IMP) is not sufficient for GVHD effects, and the regulatory function of ITK in GVHD is T cell-intrinsic [00128]
- the innate memory phenotype (IMP: CD44 hi CD122 hi and Eomes hi ) (See e.g, Huang W. Hu J, August A. Cutting edge:inate memory CD8+ T cells are distinct from homeostatic expanded CD8+ T cells and rapidly respond to primary antigenic stimulo. J.
- CD8 + T cells for CD44 hi CD122 hi and Eomes hi expression was examined, and it was observed that Itk -/- CD8 + T cells expressed substantially higher levels of CD44 hi CD122 hi and Eomes hi compared to CD8 + T cells from WT mice (FIG. 2A). It was sought to understand whether the emergence of IMP is sufficient to separate GVHD from GVL.
- WT IMP T cells were generated using a mixed-bone marrow approach in which T cell-depleted BM from WT and Itk -/- mice were mixed at a 3:1 (WT: Itk -/- ) ratio (See e.g., Huang W, et al., Cutting edge: innate memory CD8+ T cells are distinct from homeostatic expanded CD8+ T cells and rapidly respond to primary antigenic stimuli. J Immunol (2013) 190:2490–4. doi: 10.4049/jimmunol.1202988).
- Thy1.1 hosts were reconstituted with this mixture of T cell-depleted CD45.2 + WT and CD45.1 + Itk -/- BM cells, along with a control group receiving mixed CD45.2 + WT and CD45.1 + WT BM cells (FIG. 2B).
- WT BM-derived CD8 + thymocytes that develop in such mixed BM chimera acquire an IMP phenotype due to their development in the same thymus as the Itk -/- T cells, which was also observed in the experiments (FIG. 2B).
- T cells derived from WT (CD45.2 + Thy1.1-) and Itk -/- (CD45.1 + ) donor cells were sorted from the bone marrow chimeras. These sorted T cells were transplanted into irradiated BALB/c mice along with TCDBM in the allo-HSCT model as described above, and tested for their function in GVHD and GVL. Analysis of the BALB/c recipients of these different IMP CD8 + T cells indicates that WT IMP cells were not able to separate GVL and GVHD effects (Fig. 2C-G). Thus, the appearance of IMP is not sufficient to separate GVHD from GVL. [00129] More specifically, FIGS.
- FIG. 2A-2G depict the regulatory function of ITK in GVHD is T cell-intrinsic.
- FIG. 2A depicts purified WT and Itk -/- CD8 + T cells were examined for expression of CD44 prior to transplantation.
- FIG.2B depict whole bone marrow cells isolated from C57Bl/6 WT (CD45.2) and Itk -/- (CD45.1) mice were mixed in 1:3 ration WT: Itk -/- , and transplanted into irradiated Thy1.1 C57Bl/6 mice. 9-10 weeks later CD8 + T cells were sorted by CD45.2 and CD45.1 expression (donor T cells) and exclusion of Thy1.1 positive (host T cells).
- FIG. 2C depicts irradiated BALB/c mice were divided in four different groups and transplanted with the sorted T cells described in FIG.2B as follows: Group one was transplanted with 10X10 6 TCDBM alone ( TCD BM). Group two was transplanted with 10X10 6 TCD BM and 2X10 5 B-ALL-luc, (TCDBM+B-ALL luc ).
- Group three was transplanted with 10X10 6 TCDBM along with 1X10 6 purified WT CD8 + T cells and 2X10 5 B-ALL-luc ( TCD BM+B-ALL luc +WT CD45.2).
- the fourth group was transplanted 10X10 6 TCDBM along with and 1X10 6 purified Itk -/- CD8 + T cells and 2X10 5 B-ALL-luc ( TCD BM+B-ALL luc +Itk -/- CD45.1).
- These mice were monitored for leukemia cell growth using the IVIS system.
- FIG.2D the mice were monitored for survival, in FIG.2E changes in body weight, and in FIG.2F clinical score for 47 days post BMT.
- Itk -/- CD8 + and CD4 + T cells exhibit attenuated TCR signaling and an innate memory phenotype (IMP) 25 , as indicated by expression of high levels of CD44, CD122, and the key transcription factor Eomes, specifically on CD8 + T cells (FIGS. 3A-B).
- IMP innate memory phenotype
- FIGS. 3A-B To examine whether these IMP T cells from Itk -/- mice mount GVL responses, the MHC-mismatch mouse model of allo-HSCT (WT, Itk -/- ⁇ BALB/c, i.e., H2K b+ ⁇ H2K d+ ) was used.
- H2K b+ donor T cells were sorted back from recipient mice and determined their cytotoxicity against B-ALL-luc cells. It was found that these donor cells effectively killed primary leukemia cells in vitro, even in the absence of ITK (Fig.3C). Moreover, significantly increased expression of perforin in CD8 + T cells was observed from Itk -/- mice compared to T cells from WT mice, in the absence of activation (Fig.3D). The findings demonstrate that CD8 + T cells from Itk -/- mice have enhanced activation, and exert cytotoxicity against primary leukemia cells.
- IL-4 is known to upregulate Eomes in CD8 + T cells (See e.g., Weinreich, M.A., et al., T cells expressing the transcription factor PLZF regulate the development of memory-like CD8+ T cells. Nat Immunol 11, 709-716 (2010); and Pikovskaya, O. et al. Cutting Edge: Eomesodermin Is Sufficient To Direct Type 1 Innate Lymphocyte Development into the Conventional NK Lineage. J Immunol 196, 1449-1454 (2016)) were verified by comparing T cells from WT and Itk/Il4ra double KO (DKO) mice.
- DKO Itk/Il4ra double KO
- Itk/Il4ra DKO T cells cleared leukemia cells without inducing GVHD (Fig.3G).
- mice were lethally irradiated and injected intravenously with 10 ⁇ 10 6 WT T cell-depleted BM cells along with FACS-sorted CD8 + and CD4 + T cells from donor mice (WT, Itk -/- Eomes DKO). This was followed by intravenous challenge with 2 ⁇ 10 5 luciferase-expressing B-ALL-luc blast cells as described(17). Recipient animals transplanted with WT T cells cleared the tumor cells but had reduced survival and GVHD (Fig.3G-J).
- FIGS. 3A-3J it is depicted that innate memory phenotype T cells are not sufficient for GVL effect.
- FIG.3C depicts purified WT or Itk -/- T cells were transplanted into irradiated BALB/c mice, at day 7 purified T cells were sorted using H2K d , CD45.1 and CD45.2 expression. Ex vivo purified CD8 + T cells were used in cytotoxicity assay against primary leukemia target B-ALLluc+ cells at a 40:1, 20:1, or 5:1 ratio.
- FIG.3D depicts purified T cells were examined for perforin by western blot.
- FIG. 3E depicts quantitative analysis of four experiments of perforin expression by western blot with data normalized against ⁇ -Actin.
- FIG.3F depicts purified WT or Itk/Eomes DKO donor T cells were transplanted into irradiated BALB/c mice. On day 7 donor T cells were purified as described and used in an ex vivo cytotoxicity assay against B-ALL luc -cells at 5:1, 20:1, and 40:1 ratios.
- FIG.3G depicts 1X10 6 purified WT and Itk -/- Itk/Eomes DKO CD8 + T cells and 1X10 6 purified CD4 + T total of 2X10 6 mixed CD4 + and CD8 + T cells were mixed and transplanted along with 2X10 5 B-ALL-luc cells and 10X10 6 T cell-depleted bone marrow cells into irradiated BALB/c mice.
- mice Host BALB/c mice were imaged using IVIS 3 times a week. Group one received 10X10 6 T cell-depleted bone marrow alone as (TCDBM). Group two received 10X10 6 T cell- depleted bone marrow along with 2X10 5 B-ALL-luc cells ( TCD BM+B-ALL luc ). Group three were transplanted with 10X10 6 T cell-depleted bone marrow and 1X10 6 purified WT CD8 + T cells +1X10 6 CD4 + T cells and 2X10 5 B-ALL-luc cells ( TCD BM+B-ALL luc +WT CD8+CD4).
- FIG. 3G depicts the mice were monitored for survival
- FIG. 3H depicts body weight changes
- FIG. 3I depicts clinical score for 60 days post BMT.
- n 3 mice/group for BM alone
- n 5 experimental mice/group for all three group.
- the survival groups are a combination of all experiments.
- FIG. 3J depicts quantitated luciferase bioluminescence of tumor growth. Statistical analysis for survival and clinical score was performed using log- Two-way ANOVA were used for statistical analysis confirming by students t test, p values are presented. Note: Controls are na ⁇ ve mice used as negative control for bioluminescence (BLI). [00133] More specifically, referring to FIGS.
- FIGS. 8A-8G depict Itk/Il4ra DKO and WT T cells were examined for Eomes expression pre- and post- transplantation.
- FIG. 8D depicts 2X10 6 purified WT and Itk/Il4ra DKO CD8 + T and 1X10 6 purified CD4 + T cells were mixed and transplanted along with 2X10 5 primary B- ALL-luc+ cells into irradiated BALB/c mice. Recipient BALB/c mice were imaged using IVIS 3 times a week. Group one received 10X10 6 T cell-depleted bone marrow alone (TCDBM).
- TDBM T cell-depleted bone marrow alone
- Group four was transplanted 10X10 6 T cell-depleted bone marrow and 2X10 6 purified T cells (CD8 + and CD4 + ) from Itk/Il4ra DKO along with 2X10 5 B-ALL-luc cells ( TCD BM+B-ALL luc + Itk/Il4ra DKO CD8 + CD4). Recipient animals were monitored for survival, FIG.8E depicts changes in weight, and FIG. 8F depicts clinical score.
- FIG. 8G depicts Leukemia cell growth was monitored as in Figure 1, and quantitated bioluminescence is shown.
- ITK deficiency results in reduced cytokine production.
- the conditioning regimen for allo-HSCT elicits an increase in the production of inflammatory cytokines by donor T cells, known as a “cytokine storm”, and this is considered to be one of the hallmarks of GVHD pathogenesis (See e.g., D’Aveni M, Rossignol J, Coman T, Sivakumaran S, Henderson S, Manzo T, et al. G-CSF mobilizes CD34+ regulatory monocytes that inhibit graft-versushost disease. Sci Transl Med (2015) 7:281ra242. doi: 10.1126/scitranslmed.3010435).
- Blood samples were obtained from GVHD patients and healthy donors and examined the levels of serum inflammatory cytokines such as IL-33, IL-1 ⁇ , IFN ⁇ , TNF ⁇ and IL-17A. It was observed that patients with GVHD have significantly higher levels of serum proinflammatory cytokines compared to healthy controls (FIG..4A).
- cytokine production was assessed by Itk -/- CD8 and CD4 T cells in the allo-HSCT model (B6 ⁇ BALB/c), examining the levels of serum inflammatory cytokines such as IL-33, IL-1 ⁇ , IFN- ⁇ , TNF- ⁇ and IL-17A on day 7 post allotransplantation (FIG. 4B-C).
- Itk -/- donor T cells were isolate from the secondary lymphoid organs of recipients using anti-H2K b antibodies (expressed by donor C57Bl/6 cells). The cells were stimulated with anti-CD3/CD28 (FIG. 4D), or PMA/ionomycin (to bypass the proximal TCR signaling defect (See e.g., Biswas, P.S.
- Itk -/- cells produced significantly less inflammatory cytokines when stimulated via TCR/CD28 than WT cells did (FIG.4D-E).
- Purified Itk -/- CD8 + T and CD4 T cells were mixed with purified WT CD8 + or CD4 + T cells separately at a 1:1 ratio, and transplanted the mixed cells into irradiated BALB/c mice as described above.
- donor T cells were isolated from recipient mice using H2K b+ and examined for IFN- ⁇ and TNF- ⁇ expression as described above.
- the transcription factor IRF4 has been shown to play critical roles in maintaining TCR signaling, including TCR signal strength such as those regulated by ITK 31 .
- the JAK/STAT signaling pathway is also critical for the response of T cells to cytokines 32, 33 .
- expression of IRF4, JAK1, JAK2 and STAT3 was examined by purified T cells from spleens.
- FIGS.4A- 4I depicts ITK deficiency results in reduced cytokine production.
- FIG.4A depicts serum from several GVHD patients was isolated and examined for inflammatory cytokine production (IL-33, IL1 ⁇ , IFN- ⁇ and TNF- ⁇ , IL1 ⁇ and IL-17A) as determined by ELISA.
- IL-33, IL1 ⁇ , IFN- ⁇ and TNF- ⁇ , IL1 ⁇ and IL-17A 1X10 6 purified WT or Itk -/- CD8 + T or CD4 + T cells were separately transplanted with into irradiated BALB/c mice.
- recipient BALB/c were euthanized and serum cytokines (IL- 33, IL1 ⁇ , IFN- ⁇ and TNF- ⁇ and IL-17A) were measured by ELISA.
- FIGS. 4E-4F depict combined data from 3 independent experiments is shown for each experiment shown in figures.
- FIG. 4F depicts flow cytometry analysis of purified WT and Itk -/- T cells that were mixed at a 1:1 ratio for transplantation into irradiated BALB/c mice. At day 7 donor T cells were gated for expression of H-2K b , CD45.1, and CD45.2 and intracellular expression of IFN- ⁇ and TNF- ⁇ was analyzed by flow cytometry after stimulation with anti-CD3/anti-CD28.
- FIG.4G depicts purified WT or Itk -/- donor CD8 + and CD4 + T cells were transplanted into irradiated BALB/c. At day 7 donor cells were analyzed for donor T cell proliferation by examining BrdU incorporation by flow cytometry.
- FIG. 4H depicts purified WT and Itk -/- donor T cells were mixed at a 1:1 WT: Itk -/- ratio and transplanted into irradiated BALB/c mice, at day 7 donor T cells were gated for the expression of H-2K b , CD45.1, and CD45.2 and analyzed for BrDU incorporation.
- FIG.4G depicts purified WT or Itk -/- donor CD8 + and CD4 + T cells were transplanted into irradiated BALB/c. At day 7 donor cells were analyzed for donor T cell proliferation by examining BrdU incorporation by flow cytometry.
- FIG. 4H depicts purified WT and Itk -/- donor T cells were
- FIG. 9 depicts Itk -/- T cells are capable of cytokine production. Purified WT and Itk -/- T cells were transplanted into irradiated BALB/c mice. At day 7, donor T cells were gated for expression of H-2K b , CD45.2, and CD45.1, and analyzed for intracellular expression of IFN- ⁇ and TNF- ⁇ following ex vivo stimulation with PMA/ionomycin.
- FIGS.10A-10D depict quantitative analysis of JAK/STAT and IRF expression and phosphorylation. Quantitative analysis from western blots using Image Lab to normalize to ⁇ -Actin, data from 3 independent experiments.
- A Phospho and total STAT3.
- B Phospho and total JAK1.
- C Phospho and total JAK2.
- D Total IRF-4.
- ITK differentially regulates gene expression in T cells during GVHD.
- RNA sequencing analysis was employed to examine the differences in gene expression between WT and Itk -/- CD8 + T cells following allo-HSCT.
- Donor WT and Itk -/- CD8 + T cells were sort purified (using H-2K b antigen expressed by donor T cells) before and 7 days after they were transferred into irradiated BALB/c recipients, and RNA sequences was done.
- WT and Itk -/- CD8 + T cells are distinct prior to transplantation due to the enhanced IMP in the absence of ITK
- WT and Itk -/- T cells which homed to the spleen post-transplantation are similar as revealed by the fact that they clustered within a close proximity in the Principal Component Analyses (PCA) (Fig.5A). It was problematic to collect enough cells from the intestine of the Itk -/- T cell recipients, since they are deficient in homing to the intestine (Fig. 6B-D).
- PCA Principal Component Analyses
- the differentially expressed genes between WT and Itk -/- donor T cells were enriched for transcripts encoding lymphocyte homing molecules such as adhesion molecules and chemokine signaling proteins, which might contribute to the defective homing capability of Itk -/- donor T cells (FIG.5E).
- the results of critical genes that were differentially expressed were confirmed by q-RT-PCR (FIG. 5D).
- the data also revealed a critical role for ITK in regulating genes involved in T cell cytokine/cytokine receptor interaction, cell adhesion, graft-versus-host disease, allograft rejection, and chemokine signaling pathways (FIG.5E).
- FIG. 5 depicts ITK differentially regulates gene expression in T cells during GVHD.
- WT and Itk -/- CD8 + T cells were FACS sorted then transplanted into irradiated BALB/c mice.
- FIG.5A depicts principal component analysis of genes with ⁇ 2-fold change in any pairs of group combinations, with false discovery rate (FDR) ⁇ 0.05.
- WT-Pre and Itk -/- -Pre denotes cells prior to transfer
- WT-Spl denotes cells isolated from the spleen (Spl) of the recipients post-transfer.
- FIG. 5C Venn diagram of genes that are ⁇ 2-fold up- or down- regulated in the indicated comparisons, with FDR (P) ⁇ 0.05.
- FIG. 5E depicts differentially expressed genes were enriched for pathway analysis comparing WT and Itk -/- .
- FIG.5D depicts WT and Itk -/- CD8 + T cells were FACS sorted then transplanted into irradiated BALB/c mice. At day 7 post-transplant, donor T cells were sort-isolated (based on expression of H-2K b , CD3 and CD8) from host spleen and small intestine (Gut). Total RNA was isolated from sorted donor T cells were and qPCR was performed.
- GVHD involves early migration of alloreactive T cells into the target organs, followed by T cell expansion and tissue destruction. Modulation of alloreactive T cell trafficking has been suggested to play a significant role in ameliorating experimental GVHD.
- Absence of P-selectin in recipients of allogeneic bone marrow transplantation ameliorates experimental graft-versus-host disease. J Immunol (2010) 185:1912–9. doi: 10.4049/jimmunol.0903148). Therefore, the trafficking of donor T cells to GVHD target tissues was as previously described Lu SX, et al.
- both CD8 + and CD4 + T cells were tracked in allo-BMT mice by using donor CD8 + and CD4 + T cells from WT and Itk -/- mice that also express luciferase, which could be monitored by bioluminescence. It was observed that both CD8 + and CD4 + donor T cells from Itk -/- mice had significantly impaired residency in GVHD target organs, including the liver and small intestine (SI), compared to WT, despite no differences in spleen and lymph nodes (FIG. 6D).
- SI liver and small intestine
- Itk -/- T cells exhibit defective migration to target organs of GVHD, it was predicted that although Itk -/- T cells can clear leukemia cells in the blood and secondary lymphoid organs, they would not be able to kill leukemia cells that reside in tissues.
- lethally irradiated BALB/c mice were BM- transplanted together with FACS-sorted WT or Itk -/- CD8 + T cells, and challenged with subcutaneously injected B-All luc cells.
- FIG. 6 depicts ITK signaling is required for T cell migration to the GVHD target tissues.
- FIG. 6A depicts irradiated BALB/c mice were allo-HSCT-transplanted and injected with FACS-sorted WT and Itk -/- CD8 + T and CD4 + T cells mixed at a 1:1 ratio. FACS analysis of sorted T cells pre-transplant shown.
- FIG. 6B depicts at day 7 post-BMT, the spleen, liver, and small intestine (SI) were examined for donor WT and Itk -/- T cells. The ratio of WT: Itk -/- CD8 + and CD4 + T cells in the organs was determined.
- FIG. 6C depicts at day 7 post-allo-HSCT, small intestines were examined by H&E staining.
- FIG.6D depicts irradiated BALB/c mice were BM- transplanted and injected with CD8 + T CD4 + T cells from luciferase-expressing WT or Itk -/- mice.
- FIG.6E depicts on Day 7 post-allo-HSCT, donor T cells were isolated and examined for the expression of Aplnr, Cxcr5, Accr2, CCL12, CCL2, CCL5, CCr9, Ackr4, and Cmtm4 using q-RTPCR. P values were calculated using 2-way ANOVA and Student’s t test, p values are listed.
- FIG.6F depicts irradiated BALB/c mice were transplanted with C57Bl/6-derived BM and FACS-sorted WT or Itk -/- 1X10 6 CD8 + T cells, and challenged subcutaneously with 2X10 5 luciferase-expressing B-All luc cells. Recipient animals were monitored for weight changes. Group one of recipient mice was transplanted with 10X10 6 T cell-depleted bone marrow (TCD BM). The second group of recipient mice was transplanted with 10X10 6 T cell-depleted bone marrow and 2X10 5 primary B-ALL luc+ cells(TCDBM+B-ALL luc ).
- the third group of recipient mice was transplanted with 10X10 6 T cell-depleted bone marrow along with 1X10 6 T cell from WT mice along with 2X10 5 B-ALL-luc+ cells (TCDBM+B-ALL luc +WT CD8).
- the fourth group of recipient mice was transplanted with 10X10 6 T cell-depleted bone marrow and 1X10 6 T cell from Itk -/- mice along with 2X10 5 B-ALL-luc+ cells. (TCDBM+B- ALL luc + Itk -/- CD8).
- Representative bioluminescence images of leukemia cell-bearing mice on days 9, 16, 30, 38, and 47 are shown. Note: Controls are na ⁇ ve mice used as negative control for bioluminescence (BLI).
- FIG.6G depicts animals were monitored for survival over 47 days
- FIG. 6H depicts changes in weight loss
- FIG.6J depicts recipient mice were monitored for leukemia cell growth using the IVIS imaging system and quantified data is shown.
- T cells from Itk -/- mice The ability of T cells from Itk -/- mice to induce GVL without causing GVHD indicates that the ITK signaling pathway is involved in the pathogenesis of GVHD. Itk- /- T cells develop into IMP cells (CD122 + CD44 hi phenotype) in the thymus, and it is possible that such cells are responsible for the GVHD and GVL effects observed. In experiments where WT T cells developed into IMPs, it was found that they retained the capacity to induce both acute GVHD and GVL, suggesting a T cell-intrinsic function of ITK in promoting GVHD during allo-HSCT. Similarly, the cytotoxicity of Itk -/- CD8 + T cells is not dependent on the IMP.
- IMP CD8 + T cells are not responsible for distinguishing GVHD and GVL.
- Itk -/- CD8 + T cells exhibit similar or higher in vitro cytotoxicity compared to WT CD8 + T cells. This may be due to the higher levels of perforin expressed by Itk -/- T cells compared to WT T cells.
- the data also show that Itk -/- donor CD4 + and CD8 + T cells exhibit reduced expression of chemokine receptors compared to WT counterparts.
- Itk -/- donor T cells was also severely defective, reflecting the reduced expression of key chemokine receptors.
- the defective migration of Itk -/- CD8 + and CD4 + T cells likely contributes to the attenuation of GVHD, since these T cells continue to display GVL effects against leukemia cells that were injected intravenously and reside in secondary lymphoid organs.
- WT but not Itk -/- CD8 + T cells were able to inhibit leukemia cell growth when the leukemia cells were injected subcutaneously.
- the compartmentalization of T cells to secondary lymphoid organs can be an effective strategy for preventing GVHD, while leaving GVL effects against hematologic malignancies intact.
- IFN- ⁇ R signaling constitutes a major mechanism for donor T cell migration to GVHD target organs (See e.g., Choi J, Ziga ED, Ritchey J, Collins L, Prior JL, Cooper ML, et al. IFNgammaR signaling mediates alloreactive T-cell trafficking and GVHD. Blood (2012) 120:4093–103. doi: 10.1182/blood-2012-01-403196; and Choi J, Cooper ML, Alahmari B, Ritchey J, Collins L, Holt M, et al. Pharmacologic blockade of JAK1/JAK2 reduces GvHD and preserves the graft-versus-leukemia effect. PloS One (2014) 9:e109799.
- CCR2 is required for CD8-induced graft-versus-host disease. Blood (2005) 106:3322–30. doi: 10.1182/blood-2005-05-1860), which at least with CCR2 deficiency was shown to preserve the GVL effect.
- CCR5 blockade by a small molecule antagonist led to a reduction in GVHD with no significant difference in relapse rates, suggesting that blocking T cell migration to target tissues could reduce GVHD severity without compromising the beneficial GVL effect (See e.g., Terwey TH, Kim TD, Kochman AA, Hubbard VM, Lu S, Zakrzewski JL, et al.
- CCR2 is required for CD8-induced graft-versus-host disease. Blood (2005)).
- the inhibition of CXCR3 ameliorates GVHD in allo-HSCT mice.
- Activated alloreactive CD8 + T cells upregulate the expression of CX3CR1 and CXCR6 after allo-HSCT (See e.g., Duffner U, Luc B, HildebrandtGC, Teshima T,WilliamsDL, Reddy P, et al. Role of CXCR3- induced donor T-cell migration in acute GVHD. ExpHematol (2003) 31:897–902.
- CXCR6 deficiency or blockade of the CXCR3 and CXCR6 ligands attenuates GVHD (See e.g, Duffner U, Luc B, HildebrandtGC, Teshima T,Williams DL, Reddy P, et al. Role of CXCR3-induced donor T-cell migration in acute GVHD. ExpHematol (2003) 31:897–902. doi: 10.1016/S0301- 472X(03)00198-X.
- GVHD vascular endothelial growth factor
- proinflammatory cytokines exert direct effects on GVHD target tissues (See e.g., Hill GR, Crawford JM, Cooke KR, Brinson YS, Pan L, Ferrara JLM. Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood (1997) 90:3204–13.
- cytokine storm is considered one of the hallmarks of GVHD pathogenesis (See e.g., Ju XP, Xu B, Xiao ZP, Li JY, Chen L, Lu SQ, et al. Cytokine expression during acute graft-versus-host disease after allogeneic peripheral stem cell transplantation. Bone Marrow Transplant (2005) 35:1179–86. doi: 10.1038/sj.bmt.1704972), and the data showed that cytokine production was significantly reduced in mice that received Itk -/- T cells. It was also confirmed that cytokine production is T cell-intrinsic while proliferation is T cell-extrinsic.
- Example I Highlights of Example I include: 1) ITK-deficient donor T cells exhibit minimal GVHD, but maintain GVL activity; 2) ITK-deficient donor T cells exhibit significantly reduced production of inflammatory cytokines and migration to GVHD target organs; and 3) Eomesodermin (Eomes) is shown for the GVL effect.
- ITK Interleukin-2-Inducible T Cell Kinase
- Allogeneic hematopoietic stem cell transplantation is a curative therapy for hematological malignancies, due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells.
- graft-versus-host disease GVHD
- the effect of attenuating TCR-mediated SLP76:ITK interaction in GVL vs. GVHD effects was assessed after allo-HSCT.
- CD8 + and CD4 + donor T cells from mice expressing a Y145F mutation in SLP-76 did not cause GVHD, but preserved GVL effects against B-ALL cells.
- SLP76Y145FKI CD8 + and CD4 + donor T cells also showed less inflammatory cytokine production and migration to GVHD target organs.
- a novel peptide has been developed to specifically inhibit SLP76:ITK interactions, resulting in decreased phosphorylation of PLC ⁇ 1 and ERK, decreased cytokine production in human T cells, and separation of GVHD from GVL effects. Altogether, the data suggest that inhibiting SLP76:ITK interaction is a therapeutic strategy to separate GVHD from GVL effects after allo-HSCT treatment.
- Eomes flox/flox , B6.129S1, and CD4cre mice were purchased from Jackson Laboratory. Mice expressing Cre driven by the CMV promoter (CMV-Cre) were purchased from the Jackson Laboratory and crossed to ROSA26-pCAGGs-LSL-Luciferase mice (B6-luc). B6-luc mice were bred with SLP76 Y145FKI mice to create SLP76 Y145FKI luc mice. Mice aged 8-12 weeks were used, and all experiments were performed with age and sex-matched mice. Animal maintenance and experimentation were performed in accordance with the rules and guidelines set by the institutional animal care and use committees at SUNY Upstate Medical University.
- Reagents, cell lines, flow cytometry Most monoclonal antibodies for flow cytometric analysis were purchased either from eBiosciences (San Diego, CA) or Biolegend (San Diego, CA). For TCR mediated activation, we used anti-CD3 and anti- CD28. For flow cytometry analysis, we used mouse antibodies anti-CD3-FITC, anti- CD8-FITC, anti-CD4-PE anti-BrdU-APC, anti-IFN- ⁇ -APC, anti-TNF- ⁇ -PE, anti-CD122- APC, anti-CD25-BV421, and anti-FoxP3-APC.
- mice migration studies we used anti CD45.1 APC, anti-CD45.2-PE, H2KB PerCP. Anti-CD44 Pacific Blue, anti-CD122 APC and anti-Eomes PE. Human antibodies: anti-CD3-APC, anti-CD4-PE, anti-CD8- Pacific Blue, anti-TNF- ⁇ -Pe/Cy7, anti-IFN- ⁇ -APC/Cy7.
- serum ELISAs we used Biolegend LEGENDplex kits, some of which were custom ordered to detect both mouse and human cytokines.
- luciferin was purchased from Gold Bio (St Louis, MO). To exclude dead cells from analyses, we used LIVE/DEAD Fixable Aqua Dead Cell staining.
- B-ALL Primary mouse B-cell acute lymphoblastic leukemia (B-ALL) blasts and primary cells (Cheng et al., 2016) were transduced with luciferase, and cultured as described previously (Edinger et al., 2003). B-ALL was chosen for this model because (1) these cells are syngeneic with BALB/c mice and allogeneic to C57Bl/6 mice, and (2) B-ALL was selected to be more related to human disease (Cheng et al., 2016).
- GVHD and GVL studies Recipient BALB/c mice (MHC haplotype d) as recipients were lethally irradiated with 800 cGy total in two split doses of 400cGy. Bone marrow cells were harvested from mouse legs, and total bone marrow cells were incubated with CD90.2 beads, using 100ul of beads per mouse according to manufacturer protocols. NK cells were depleted and by DX5 beads, and CD122 + cells with anti PE beads.
- Recipient mice were injected intravenously with 10X10 6 T cell- depleted bone marrow ( TCD BM) cells, with or without donor T cells, either 1X10 6 or 2X10 6 FACS-sorted CD8 + , CD4 + T cells, or CD8 + and CD4 + cells mixed at a 1:1 ratio from WT or SLP76 Y145FKI mice.
- TCD BM T cell- depleted bone marrow
- B-ALL primary B cell acute lymphoblastic leukemia
- luciferase 2X10 5 luciferase-expressing B-ALL-luc cells
- All recipient animals were examined for tumor burden twice a week from the time of challenge with B-ALL luc injection until 70 days post- transplant, using bioluminescence imaging with the IVIS 50 and IVIS 200 imaging systems (Xenogen) as previously described (Contag and Bachmann, 2002). Each mouse was injected with 10 ⁇ g/g body weight of luciferin and imaged for 1 minute.
- Serum was examined for IL-33, IL-1a, IFN-g, TNF-a and IL-17A by multiplex cytokine assays (Biolegend LEGENDplex).
- splenocytes were processed to obtain single cells, and T cells were stimulated with anti-CD3 and anti-CD28 for 6 hours in the presence of brefeldin A (10mM). After 6 hours, stimulated cells were stained for surface markers and stained intracellularly for cytokines (IFN-g and TNF-a).
- cytokines IFN-g and TNF-a
- Proliferation Assays For detection of BrdU, transplanted mice (as described above) were given BrdU with an initial bolus of BrdU (2 mg per 200 ⁇ l intraperitoneally) and drinking water containing BrdU (1 mg/ml) for 2 days. BrDU incorporation was performed using a BrDU kit (Invitrogen) according to the manufacturer’s instructions.
- Cytotoxicity assays For cytotoxicity assays, luciferase-expressing A20 and B-ALL cells (both allogenic to BALB/c) were seeded in 96-well flat bottom plates at a concentration of 3X10 5 cells/ml.
- D-firefly luciferin potassium salt 75 mg/ml; Caliper Hopkinton, MA
- luciferin potassium salt 75 mg/ml; Caliper Hopkinton, MA
- ex vivo effector cells (MACS-sorted or FACS-sorted CD8 + T cells from bone marrow-transplanted mice) were added at 40:1, 20:1, and 10:1 effector-to-target (E:T) ratios and incubated at 37°C for 4 hours.
- Bioluminescence in relative luciferase units (RLU) was then measured for 1 minute. Cells treated with 1% Nonidet P-40 were used as a measure of maximal killing. Target cells incubated without effector cells were used to measure spontaneous death.
- % specific lysis 100 ⁇ (spontaneous death RLU–test RLU)/(spontaneous death RLU– maximal killing RLU)(Karimi et al., 2014).
- % specific lysis 100 ⁇ (spontaneous death RLU–test RLU)/(spontaneous death RLU– maximal killing RLU)(Karimi et al., 2014).
- Tissue Imaging Allo-HSCT was performed with 10X10 6 WT T cell- depleted BM cells and 1X10 6 FACS-sorted CD8 + or 1X10 6 FACS-sorted CD4 + T cells (from B6-luc or SLP76Y145FKI luc mice) and bioluminescence imaging of tissues was performed as previously described (Beilhack et al., 2005).
- luciferin 10 ⁇ g/g body weight
- selected tissues were prepared and imaged for 5 minutes. Imaging data were analyzed and quantified with Living Image Software (Xenogen) and Igor Pro (Wave Metrics, Lake Oswego, OR).
- Livers were perfused with PBS, dissociated, and filtered with a 70mm filter.
- the small intestines were washed in media, shaken in strip buffer at 37°C for 30 minutes to remove the epithelial cells, and then washed, before digesting with collagenase D (100 mg/ml) and DNase (1mg/ml) for 30 minutes in 37°C, and followed by filtering with a 70 mm filter. Lymphocytes from the liver and intestines were further enriched using a 40% Percoll gradient.
- T cells were analyzed for CD8 + T cells and CD4 + T cells and presence of H2K b , CD45.1 + and CD45.2 + (to identify the transferred T cell populations) by flow cytometry, but we excluded any bone marrow-derived T cells (Thy1.1 + ).
- Western blotting For protein analysis, T cells were either nonstimulated, or stimulated with anti-CD3 and anti-CD28 for 24 hours overnight, and were lysed with freshly prepared lysis buffer (RIPA Buffer(Fisher Scientific cat#PI89900) + cOmplete Protease Inhibitor Cocktail (Sigma-Aldrich; cat# 11697498001)) and centrifuged for 10 minutes at 14000rpm at 4°C.
- qPCR assay Post-transplanted donor CD8 + and CD4 + T cells from C57Bl/6 mice (MHC haplotype b) were FACS sorted from recipient mice on H2K b markers, and total RNA was isolated from T cells using the RNeasy kit from Qiagen (Germantown, MD). cDNA was made from total RNA using a cDNA synthesis kit (Invitrogen).
- SLP76pTYR Peptide To generate a molecule that specifically inhibits the interaction between pY145 of SLP76 and the SH2 domain of ITK, a peptide was designed based on the amino acid sequence of SLP76 from N132 to A155, which contains a phosphorylated tyrosine residue at Y145 (FIG. 17).
- SLP76pTYR peptide (FIG. 17). Both SLP76pTYR peptide (FITC Dye - 132 NEEEEAPVEDDADpYEPPPSNDEEA 155 -TAT) (SEQ ID NO:5) and non-specific control peptide (FITC Dye-IIMTTTTNNKKSSRRVVVVAAAADD-TAT) (SEQ ID NO:6) were synthesized by Genscript Inc (Piscataway, NJ).
- T cells were isolated from WT mice, and T cells were generated from splenocytes as previously described (Baker et al., 2001). Briefly, T cells were isolated from splenocytes using MACS beads (Miltenyi Biotec), then cultured in complete RPMI media (3X10 6 cells/mL) and plated on anti-CD3 (2.5 mg/ml; Biolegend; cat#100202) and anti-CD28 (2.5 mg/ml; Biolegend; cat#102116) antibody-coated tissue culture plates until otherwise specified.
- T cells were incubated with SLP76pTYR, control peptide or vehicle alone at different concentrations ranging from 100 ng/ml to 1 mg/ml in the presence of 4 mg/ml of protamine sulfate.
- Protamine sulfate significantly increased peptide delivery into primary cells. Within 60 minutes, it was observed that peptides were inside the cells. Cells were cultured for 5 minutes prior to investigating signaling changes.
- FITC Fluorescence Activated Cells were examined for the presence of FITC by microscopy using a Leica DMi8 microscope equipped with an infinity total internal reflection fluorescence (TIRF) and DIC modules, a Lumencor SOLA SE II light box, a 150 mW 488 (GFP) laser and filter cube, a 100x/1.47 NA objective, and an Andor iXon Life 897 EMCCD camera. FITC expression was confirmed by flow cytometry as well. Cells were lysed and used in Western blots. [00165] Human Samples: According to our IRB protocol (1140566-4), blood samples were obtained by vein puncture, and T cells were isolated from peripheral blood mononuclear cells (PBMC) of regular healthy donors.
- PBMC peripheral blood mononuclear cells
- T cells were isolated from patient and healthy donor samples by Ficoll-Hypaque density centrifugation. The final product was resuspended at 3X10 6 cells/ml in media and stimulated with OKT-3/anti- CD3 (2.5 mg/ml; Ortho Bio-Tech) and anti-human CD28 (2.5 mg/ml; Biolegend; cat#302902) presence of 4 mg/ml of protamine sulfate and 1 mg/ml SLP76pTYR or vehicle for five minutes. T cell lysates were used in western blot analysis.
- Transducing primary T cells with SLP76pTYR viruses were generated that specifically express SLP76pTYR; the sequences encoding SLP76pTYR were cloned as a fusion protein with pCherry (plasmid depicted in FIG. 22) ordered through Integrated DNA Technology (IDT). The insert was cloned into a pQCX-I-X retroviral vector between MLU1 and Xho1 restriction sites, and the insert was confirmed by digestion and sequencing. To generate retroviral supernatants, Phoenix packaging cells were plated in 60 cm 2 dishes and transfected with 20 ⁇ g of the vector using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol.
- IDT Integrated DNA Technology
- the medium was changed after 8–12 hours, and viral supernatants were harvested after 24–36 hours. Concentrated viral supernatants were re-suspended in MDM media (Invitrogen) and used to transduce primary T cells in the presence of protamine sulfate (4 ⁇ g/ml) to enhance transduction efficiency. T cells were transduced with viruses containing either SLP76pTYR-pCherry or empty plasmid for 24 hours and then injected into mice. [00167] Statistics. All numerical data are reported as means with standard deviation. Data were analyzed for significance with GraphPad Prism. Differences were determined using one-way or two-way ANOVA and Tukey’s multiple comparisons tests, or with a student’s t-test when necessary.
- GVHD graft-vs-host disease
- Mature T cells in the graft facilitate stem cell engraftment and, most importantly, ensure the therapeutic graft-versus-leukemia (GVL) effect (Breems and Lowenberg, 2005, Tugues et al., 2018).
- GVHD graft-versus-host disease
- standard immunosuppressive therapy for GVHD is not optimal, because it leaves patients susceptible to opportunistic infections such as Cytomegalovirus (CMV) and relapse of the malignancy being treated (Bleakley et al., 2012) (Ferrara, 2014).
- CMV Cytomegalovirus
- the pathophysiology of GVHD depends upon interactions between donor T cells and host antigen-presenting cells (APCs). T cell receptor (TCR)-mediated activation of donor T cells by APCs is critical for both GVHD and GVL effects (Guinan et al., 1999) .
- APCs host antigen-presenting cells
- GVHD can be treated by interfering with T-cell activation and proliferation using calcineurin inhibitors (cyclosporine, tacrolimus), mTOR inhibitors (sirolimus), and antiproliferative agents (methotrexate, cyclophosphamide, or mycophenolate) (Reddy and Ferrara, 2008, Baxter and Hodgkin, 2002) .
- calcineurin inhibitors cyclosporine, tacrolimus
- mTOR inhibitors sirolimus
- antiproliferative agents metalhotrexate, cyclophosphamide, or mycophenolate
- T cell signaling requires a multimolecular proximal signaling complex, which includes adapter proteins such as SLP76 (Koretzky et al., 2006, Kambayashi et al., 2009). SLP is involved in phosphorylation of phospholipase C-gamma isoforms by ITK in T cells (Su et al., 1999). IL-2-inducible T-cell kinase (ITK) is a critical mediator of T cell receptor (TCR) signaling (Bunnell et al., 2000).
- TCR T cell receptor
- SLP-76 activates ITK through its N-terminal tyrosine at the position Y145 (Bogin et al., 2007, Jordan et al., 2006).
- the tyrosine residue 145 (Y145) of SLP76 binds to and activates the Tec family tyrosine kinase ITK (Bogin et al., 2007).
- a YàF mutation at Y145 of SLP76 leads to defective TCR-mediated ITK activation (Jordan et al., 2006).
- the SLP76 Y145 and ITK interaction is involved in signaling pathways that lead to cytokine production by T cell populations, as well as in regulating the development of a distinct, innate-type cytokine-producing T cell population in the thymus (Atherly et al., 2006), referred to as innate memory phenotype (IMP) T cells.
- CD4 + and CD8 + T cells from SLP76Y145FKI mice express significantly higher CD122, CD44, and Eomes compared to T cells from WT mice on a basal, unstimulated level (Mammadli et al., 2020) .
- CD4 + and CD8 + T cells with CD44 low are considered naive cells.
- CD4 + and CD8 + T cells with CD44 high are considered antigen-experienced and activated cells.
- Both CD4 + and CD8 + T cells from SLP76Y145F and ITK-deficient mice express a higher proportion of cells with CD44hi and CD122hi (Mammadli et al., 2020).
- CD4 + and CD8 + T cells with CD44 hi and CD122 hi from SLP76Y145FKI and ITK deficient mice arise in the thymus during development, unlike memory CD44 hi CD4 + and CD8 + T cells that mainly arise in the periphery of WT mice in response to foreign antigens or because of homeostatic proliferation (Mammadli et al., 2020) .
- Experimental studies in CD44 hi and CD44 lo cells arising in the periphery of WT mice show conflicting results (Dutt et al., 2011, Zheng et al., 2009, Loschi et al., 2015, Anderson, 2003, Huang et al., 2019).
- WT CD8 + CD44 lo T cells induce severe GVHD, and that while WT CD8 + CD44 hi T cells induce less GVHD, as has been reported, they eventually cause GVHD (Zhang et al., 2005, Huang et al., 2019).
- SLP76Y145FKI CD8 + CD44 lo T cells are much less likely to induce GVHD, and SLP76Y145FKI CD8 + CD44 hi T cells do not cause GVHD, but maintain a significant GVL effect (Mammadli et al., 2020).
- Both CD8 + and CD4 + SLP76Y145FKI T cells exhibit attenuated TCR signaling and an innate memory phenotype (IMP) as indicated by expression of high levels of CD44 and CD122, and CD8 + SLP76Y145FKI T cells also express higher levels of the transcription factor Eomes (Huang et al., 2014, Carty et al., 2014).
- IMP innate memory phenotype
- the data suggest that IMP phenotype may not be enough to separate the wanted effect of GVL from the unwanted GVHD effect. It is also shown that disruption of the SLP76Y145/ITK interaction allows T cells to differentiate GVHD from GVL effects.
- SLP76145pTYR a novel peptide inhibitor that disrupts the interaction between SLP76 and ITK. It is shown that SLP76145pTYR specifically inhibits the phosphorylation of ITK and downstream signaling molecules, including PLC ⁇ 1 and ERK, in both human and mouse T cells (Kim et al., 2009).
- ITK is differentially required for GVHD and GVL (Mammadli et al., 2020). Therefore, it was tested whether this distinction depends on the interaction of ITK with SLP76.
- T cell signaling requires a multimolecular proximal signaling complex that includes adapter proteins such as SLP76 (Koretzky et al., 2006, Kambayashi et al., 2009).
- SLP76 is phosphorylated on tyrosine residue 145 (Y145), it binds to and activates the Tec family tyrosine kinase ITK (Bogin et al., 2007).
- a Y ⁇ F mutation at Y145 (such as substitution characterized as Y145F) of SLP- 76 leads to defective TCR-mediated ITK activation(Jordan et al., 2006), with effects on signaling pathways that lead to cytokine production by T cell populations.
- SLP76 Given the role of SLP76 in regulating ITK signaling downstream of the T cell receptor, it was tested whether GVL effects would remain intact when allo-BMT was performed with T cells from SLP76 Y145FKI mice.
- TCD BM T cell-depleted bone marrow
- B6.PL- Thy1a/CyJ mice donor T cells from C57BL/6 (B6) WT or SLP76Y145FKI mice (MHC haplotype b), and lethally irradiated BALB/c (MHC haplotype d) mice as recipients.
- Recipient mice were injected intravenously with 10X10 6 wild-type (WT) TCDBM cells along with 2 ⁇ 10 6 FACS-sorted donor T cells (1X10 6 CD8 + and 1X10 6 CD4 + ).
- B-ALL B-cell acute lymphoblastic leukemia
- B-ALL 2X10 5 luciferase-expressing primary B-cell acute lymphoblastic leukemia (B-ALL)-luc blast cells as previously described (Cheng et al., 2016) were mixed with TCDBM and CD4 and CD8 T cells, and intravenously injected into recipient BALB/c mice by tail vein.
- B-ALL is a primary B cell acute lymphoblastic leukemia, syngeneic to BALB/c mice and allogeneic to C57BL/6 (B6) mice.
- B-ALL cells were mixed with donor T cells and TCD BM right before injection. Recipient BALB/c mice were monitored for cancer cell growth using IVIS bioluminescence imaging for over 60 days (FIG. 13A).
- mice transplanted with CD4 + and CD8 + T cells from either WT or SLP76Y145FKI mice suffered from GVHD
- Donor CD8 + T cells are more potent than CD4 + T cells in mediating GVL effects, but both CD4 + and CD8 + T cells mediate severe GVHD in mice and humans (Amir et al., 2012, Yu et al., 2006, Wu et al., 2013).
- CD4 + T cell-intrinsic SLP76:ITK signaling might be sufficient to induce GVHD, we repeated the same experiments using purified CD4 + T cells from either WT or SLP76Y145FKI mice in the MHC- mismatch mouse model of allo-HSCT (B6 ⁇ BALB/c) (FIG. 20A-C).
- FIGS.13A-13E depict disruption of ITK:SLP76 Y145 signaling allows tumor clearance without inducing GVHD.1X10 6 purified CD8 + T cells and 1X10 6 purified CD4 + T cells from WT or SLP76 Y145FKI mice were mixed at a 1:1 ratio and transplanted with 2X10 5 B-ALL cells and 10X10 6 T cell-depleted bone marrow ( TCD BM) cells transplanted into irradiated BALB/c mice. Host BALB/c mice were imaged using the IVIS 50 system three times a week. (FIG. 13A) Group one received 10X10 6 T cell depleted bone marrow cells ( TCD BM) only.
- mice are used as negative controls while imaging other groups that have luciferase-expressing primary leukemia cells.
- Group two received 10X10 6 TCD BM with 2X10 5 B-ALL-luc cells (TCDBM+B-ALL luc).
- the third group was transplanted with 10X10 6 TCDBM cells and 1X10 6 purified WT CD8 + and 1X10 6 CD4 + T cells (1:1 ratio) along with 2X10 5 B-ALL - luc cells (TCDBM+B-ALL-luc+WT CD8 + and CD4 + ).
- FIG. 20A-20C depict SLP76 Y145FKI CD4 + T cells exhibit attenuated induction of GVHD compared to WT T cells.10X10 6 T cell-depleted bone marrow cells and 1X10 6 purified WT or SLP76 Y154FKI CD4 + T cells were transplanted into irradiated BALB/c mice.
- Recipient mice were injected with 10X10 6 WT C57Bl/6 TCD BM cells, with or without 1X10 6 FACS-sorted CD8 + CD44 lo CD122 lo or CD8 + CD44 hi CD122 hi (IMP) T cells from WT or SLP76Y145FKI mice (Fig.21B).
- Recipient mice were challenged with 1X10 5 primary B-ALL-luc (Cheng et al., 2016) tumor cells and monitored for survival, weight changes, clinical score, and tumor burden (monitored by bioluminescence imaging twice a week) for at least 60 days (Fig.21B).
- FIGS.21A-21F depict the innate memory phenotype of CD8 + T cells does not separate GVHD and GVL effects.
- FIG.21A Purified T cells from WT and SLP76Y145FKI mice were examined for expression of CD44 pre- and post-sort.
- FIG.21B All recipient BALB/c mice were lethally irradiated and divided into six different groups.
- Group one was transplanted with 10X10 6 TCD BM.
- Group 2 was transplanted with 10X10 6 TCDBM and 1X10 5 B-ALL-luc.
- Group 3 was transplanted with 10X10 6 TCD BM along with 1X10 6 purified WT CD8 + CD44 lo T cells and 1X10 5 B-ALL- luc.
- Group 4 was transplanted with 10X10 6 TCDBM along with 1X10 6 purified WT CD8 + CD44 hi T cells, and 1X10 5 B-ALL-luc.
- Group 5 was transplanted 10X10 6 TCD BM along with 1X10 6 purified SLP76Y145FKI CD8 + CD44 lo T cells and 1X10 5 B-ALL-luc.
- Group 6 was transplanted with 10X10 6 TCD BM and 1X10 6 purified SLP76Y145FKI CD8 + CD44 hi T cells and 1X10 5 B-ALL-luc. These mice were monitored for tumor growth using the IVIS 50 system.
- FIG.21F Quantitated luciferase bioluminescence of luciferase- expressing B-ALL cells.
- CD4 + or CD8 + T cells with attenuated TCR signaling produce inflammatory cytokines similar to CD4 + or CD8 + T cells from WT mice
- 1X10 6 CD4 + or CD8 + T cells were transplanted in separate experiments from either WT mice or SLP76Y145FKI mice to irradiated BALB/c mice as recipients.
- recipient BALB/c mice were sacrificed, and serum was obtained and assessed for levels of the proinflammatory cytokines IL-33, IL-1 ⁇ , IFN- ⁇ , TNF- ⁇ , and IL-17A by multiplex ELISA (Fig. 14A-B).
- recipient animals transplanted with CD4 + or CD8 + T cells had significantly less production of proinflammatory cytokines compared to recipient mice transplanted with CD4 + or CD8 + T cells from WT mice (Fig.14A-B).
- spleen cells were restimulated from recipient mice that were transplanted with CD4 + or CD8 + T cells from WT or SLP76Y145FKI mice in separate experiments.
- Spleen cells were restimulated with anti-CD3 and anti-CD28 in the presence of Brefeldin A, and donor CD4 + and CD8 + T cells were gated by flow cytometry using anti-H2K b antibodies (expressed by donor cells), anti-CD3, anti-CD4, and anti-CD8. It was observed that donor CD4 + and CD8 + T cells from SLP76Y145FKI mice produced significantly less IFN- ⁇ and TNF- ⁇ compared to donor CD4 + and CD8 + T cells from WT mice. Thus, these data confirm that the changes in proinflammatory cytokine serum levels result from changes in production by the donor T cells, and that T cells from TCR-attenuated mice produce less inflammatory cytokines (Fig.14C-E).
- SLP76Y145FKI T cells are capable of producing cytokines in general
- spleen cells from recipient mice transplanted with either WT or SLP76Y145FKI T cells were stimulated with PMA/ionomycin (to bypass the proximal signaling defect (Fig.23)), or left unstimulated for 6 hours in the presence of Brefeldin A, followed by the analysis of IFN- ⁇ and TNF- ⁇ production.
- SLP76Y145FKI T cells were capable of producing IFN- ⁇ and TNF- ⁇ when T cell signaling was bypassed by re-stimulation with PMA and ionomycin (Fig.
- SLP76Y145FKI donor T cells were mixed purified with purified WT CD8 + or CD4 + T cells separately at a 1:1 ratio, and transplanted the mixed cells into irradiated BALB/c mice as described above.
- the congenic markers CD45.1 WT C57BL/6) and CD45.2 (SLP76Y145FKI) were used to distinguish donor cells from the different strains of mice within the same recipient.
- donor T cells were isolated from recipient mice using flow cytometry (anti-H2Kb) and examined for IFN- ⁇ and TNF- ⁇ expression as described above. It was found that WT donor CD8 + and CD4 + T cells (CD45.1) produced higher levels of inflammatory cytokines than SLP76Y145FKI donor CD8 + and CD4 + T cells (CD45.2), suggesting that the reduced cytokine production observed by SLP76Y145FKI donor T cells is T cell-intrinsic (Fig.14F). [00178] More specifically, FIGS.
- FIG.14G Purified CD8 + or CD4 + WT or SLP76Y145FKI donor T cells were transplanted into irradiated BALB/c mice. On day seven, donor T cells were analyzed for donor CD8 + or CD4 + T cell proliferation by examining BrdU incorporation by flow cytometry.
- FIG.14H Purified CD8 + or CD4 + T cells from WT or SLP76Y145FKI mice were mixed at a 1:1 WT:SLP76Y145FKI ratio and transplanted into irradiated BALB/c mice. At day 7, splenic donor T cells were gated for the expression of H-2K b , CD45.1, and CD45.2, and analyzed for BrdU incorporation.
- FIG.23 depicts SLP76Y145FKI T cells are capable of cytokine production.
- Purified T cells from WT and SLP76Y145FKI C57Bl/6 mice were transplanted into irradiated BALB/c mice (MHC haplotype d) as recipients.
- donor T cells were gated for expression of H-2K b , CD45.2, and CD45.1, and analyzed for intracellular expression of IFN- ⁇ and TNF- ⁇ following ex vivo stimulation with PMA/ionomycin.
- Data from several experiments were combined, and statistical analysis performed using two-way ANOVA and Student’s t-test, with p values presented.
- SLP76Y145FKI donor T cells proliferated similarly to WT donor CD4 + and CD8 + T cells.
- Lethally irradiated recipient BALB/c mice were transplanted as mentioned above, with either WT or SLP76Y145FKI donor CD4 + or CD8 + T cells.
- Recipient mice were injected with BrdU as described, and seven days post-allotransplantation, recipient mice were sacrificed and examined for proliferation by BrdU incorporation.
- SLP76Y145FKI donor T cells showed reduced proliferation compared to WT donor T cells Fig.14G.
- FIGS. 15A-15H depict eomes is required for cytotoxicity and GVL effect by both WT and SLP76Y145FKI T cells.
- FIGS. 15A-B Purified WT and SLP76Y145FKI CD8 + and CD4 + T cells were examined for expression of CD44, CD122, and Eomes by flow cytometry.
- FIG. 15C Purified donor CD8 + T cells from either WT or SLP76 Y145FKI Eomes-sufficient, Eomes-deficient, or Eomes- flox control mice were transplanted into irradiated BALB/c (MHC haplotype d) mice. On day seven, donor T cells were purified as described and used in an ex vivo cytotoxicity assay against B-ALL-luc cells at 40:1, 20:1, and 10:1 ratios. (FIG.
- Group three was transplanted with 10X10 6 TCDBM and 1X10 6 purified WT CD8 + T cells +1X10 6 CD4 + T cells, and 2X10 5 B-ALL-luc cells (TCDBM+B-ALL luc +WT CD8+CD4).
- Group four received 10X10 6 TCDBM and 1X10 6 purified CD8 + T cells +1X10 6 CD4 + T cells from SLP76 Y145FKI Eomes-sufficient mice along with 2X10 5 B-ALL-luc cells ( TCD BM+B ALL luc +SLP75Y145FKI CD8+CD4).
- TCDBM+B-ALL luc + SLP75Y145FKI Eomes-cKO CD8+CD4 Group six received 10X10 6 TCDBM and 1X10 6 CD8 + T cells +1X10 6 CD4 + purified T cells from WT Eomes-deficient mice along with 2X10 5 B-ALL-
- FIG.15H Luciferase bioluminescence of tumor growth was quantified. Statistical analysis for survival and the clinical score was performed using log calculation. Two-way ANOVA was used for statistical analysis and results were confirmed by students t-test, p values are presented.
- mice Lethally irradiated BALB/c mice were injected intravenously with 10X10 6 WT T cell-depleted BM cells along with 1X10 6 FACS-sorted CD8 + and CD4 + T cells from either WT mice or SLP76Y145FKI Eomes flox/flox (SLP76Y145FKI Eomes cKO) mice, with or without CD4cre (to delete Eomes specifically in CD4 + and CD8 + T cells), along with 2X10 5 luciferase-expressing B-ALL- luc blast cells as described (Cheng et al., 2016) . Recipient animals transplanted with WT T cells cleared the tumor cells but developed acute GVHD (Fig. 15D).
- the bioluminescence data were analyzed and quantified with Living Image Software (Xenogen) and Igor Pro (Wave Metrics, Lake Oswego, OR) (Fig. 15H).
- recipient animals transplanted with Eomes deficient (Eomes flox/flox mice with CD4cre, called SLP76Y145FKI Eomes cKO) T cells could not clear the tumor, and all died from tumor burden.
- Recipient animals transplanted with WT Eomes deficient (Eomes flox/flox mice with CD4cre, called WT Eomes cKO) T cells developed severe GVHD and were also unable to clear transplanted leukemia cells.
- FIGS. 24A and 24B depict eomes deletion on CD8 + and CD4 + T cells.
- FIG.24A Purified donor CD8 + and CD4 + T cells from either WT or WT Eomes-deficient (Eomes cKO) mice on a C57Bl/6 background were examined for Eomes expression.
- FIG. 24B Quantitative analysis from flow cytometry data of several experiments. For statistical analysis a two-way ANOVA and student’s t test was used, p values are presented.
- SLP76Y145/ITK signaling is needed for T cell migration to the GVHD target tissues.
- GVHD involves early migration of alloreactive donor T cells into the target organs, followed by T cell expansion and tissue destruction (Ferrara, 2014). Modulation of alloreactive T cell trafficking has been suggested to play a significant role in ameliorating experimental GVHD (Lu et al., 2010). Therefore, the trafficking of donor T cells to GVHD target tissues was examined, as previously described (Lu et al., 2010) .
- Irradiated BALB/c recipient mice were injected with CD8 + and CD4 + T cells from C57Bl/6 background SLP76Y145FKI (CD45.2 + ) and WT B6LY5 (CD45.1 + ) mice mixed at a 1:1 ratio (Fig. 16A), and seven days post-transplantation, recipient mice were examined for the presence of donor CD8 + and CD4 + T cells in the spleen, lymph nodes, liver, and small intestines. While the WT: SLP76Y145FKI T cell ratio for both CD8 + and CD4 + cells remained approximately 1:1 in the spleen and lymph nodes (Fig.
- donor CD4 + and CD8 + T cells were FACS sorted from the recipient using H2K b expression. Purified donor CD4 + and CD8 + T cells were examined for chemokine receptor expression by qPCR.
- chemokine receptors and other molecules that play a critical role in T cell migration were significantly reduced in SLP76Y145FKI CD8 + and CD4 + T cells at day 7 post- transplantation compared to WT CD8 + and CD4 + T cells (Fig.16E-F).
- both CD8 + and CD4 + T cells were tracked in allo-BMT mice by using donor CD8 + and CD4 + T cells from WT and SLP76Y145FKI mice that also express luciferase, which could be monitored by bioluminescence (Negrin and Contag, 2006). It was observed that both CD8 + and CD4 + donor T cells from SLP76Y145FKI mice had significantly impaired residency in GVHD target organs - including the liver and small intestine (SI) - compared to WT, despite no differences in spleen and lymph nodes (Fig.16G).
- SI liver and small intestine
- FIGS. 16A-16G depict SLP76Y145/ITK signaling is required for T cell migration to the GVHD target tissues.
- FIG.16A Irradiated BALB/c mice were allo-transplanted and injected with FACS-sorted CD8 + or CD4 + T cells mixed at a 1:1 ratio from WT B6.SJL (Ly5 CD45.1) and WT C57B16 (CD45.2) mice.
- FACS-sorted CD8 + or CD4 + T cells from B6.SJL (Ly5 CD45.1) and SLP75Y145FKI (C57B16 in background, CD45.2) mice at a 1:1 ratio.
- FACS analysis of sorted T cells pre-transplant is shown.
- FIGS.16B-C On day seven post- transplantation, the spleen, liver, and small intestine (SI) from recipients were examined for donor CD4 and CD8 + T cells by H2K b+ CD45.1 + (LY5) or CD45.2 + (B6). We also examined donor CD4 or CD8 + T cells from WT mice by H2Kb + CD45.1 + , and from SLP75Y145FKI mice by H2Kb + and CD45.2 + . The ratio of WT: SLP75Y145FKI CD8 + and CD4 + T cells in the organs was determined. (D) We transplanted either CD4 + or CD8 + T cells separately into irradiated BALB/c mice, in separate experiments.
- Irradiated BALB/c mice were BM-transplanted and injected with CD8 + T cells and CD4 + T cells from luciferase-expressing WT or SLP75Y145FKI mice (C57Bl/6 background).
- recipient BALB/c mice were injected with D-luciferin. Spleen, lymph nodes, liver, and small intestine were examined for donor CD8 + T cells or CD4 + T cells by luciferase expression.
- FIGS. 25A and 25B depict quantitative analysis of donor T cells in secondary lymphoid organs and GVHD target organs.
- FIGS. 25A and 25B depict quantitative analysis of donor T cells in secondary lymphoid organs and GVHD target organs.
- FIG. 25A Quantitative analysis from flow cytometry data. CD8 + and CD4 + T cells from WT and SLP76Y145FKI C57Bl/6 mice were transplanted into BALB/c mice (MHC haplotype d) as recipients. In several experiments, donor CD4 + and CD8 + T cells were analyzed for migration in the secondary lymphoid organs spleens and lymph nodes.
- FIGS. 26A and 26B depict quantitative analysis of tissue bioluminescence imaging (BLI).
- Novel peptide inhibitor SLP76pTYR specifically targets ITK signaling and enhances Treg cell development. Since T cells from SLP76Y145FKI mice can separate GVHD from GVL, we sought to determine whether disruption of ITK signaling with pharmacological agents would have a similar effect.
- the SH2 domain of ITK contains a complementary electrostatic surface, because the phosphotyrosine binding pocket, as well as the surrounding surface groove, is highly positively charged, suggesting that electrostatics most likely will play a vital role in this interaction (Fig.17B).
- the SLP76pTYR construct consists of two components (Fig. 17C): (i) amino acid residues 132 to 155 of SLP76 with phosphorylated tyrosine at position 145 and (ii) a TAT peptide sequence (GRKKRRQRRRPQ) (SEQ ID NO:3) (such as viral sequences) for cell membrane penetration (i.e.
- T cells were cultured with a FITC-conjugated SLP76pTYR peptide, vehicle, or nonspecific peptide, and examined the cells for FITC uptake using microscopy and flow cytometry. It was observed that significant numbers of cells were positive for FITC following treatment with the SLP76pTYR peptide (Fig. 17D-F). The peptide was localized in specific locations in the cell as observed by imaging the cells in a single focal plane near the cover glass.
- ITK deficiency is known to enhance the development of regulatory T cells (Tregs) (Elmore et al., 2020, Owen et al., 2019) so the peptide inhibitor was tested to determine whether inhibition of the ITK:SLP76Y145 interactions using SLP76pTYR would induce Tregs.
- Tregs regulatory T cells
- the frequency of Tregs was examined by expression of CD4 + and FoxP3 + (Fig.18A).
- mice T cells were stimulated with anti-CD3 (Sugie et al., 2004) in the presence of either SLP76pTYR, nonspecific peptide, or vehicle alone for 5 to 24 hours, and cells were harvested and examined for the presence of Tregs (CD4 + CD25 + FoxP3 + ). Significantly enhanced differentiation of Treg cells in T cell cultures treated with SLP76pTYR peptide were observed compared to vehicle alone or nonspecific peptide (Fig.18B-C). Next, mouse T cells were stimulated with anti-CD3 and anti-CD28 for 5 minutes, in the presence of SLP76pTYR or vehicle alone, and observed a reduction in ITK phosphorylation.
- SLP76pTYR peptide has an impact on signaling pathways downstream of SLP76 in both mouse and human T cells.
- SLP76pTYR peptide exhibited minimal off-target effects against other kinases, including PI3K and AKT (Fig.28A-H). It is possible that PI3K and AKT lie downstream of ITK, but that the specific pathways affected by the disruption of the SLP76:ITK interaction does not affect the activation of PI3K and AKT.
- Fig. 18E the ability of SLP76pTYR peptide to affect the production of proinflammatory cytokines in human PBMCs was investigated.
- FIGS.17A-17F depict development of a novel peptide that disrupts the interaction between SLP76 and ITK.
- FIG.17A NMR spectroscopy structure of murine ITK SH2 domain showing its complex with a peptide containing a pTyr residue (PDB code:2ETZ) that was previously solved by Pletneva et al.
- the SH2 domain is rendered in surface representation (wheat), while the peptide derived from residues 143-148 of SLP76 with a sequence ( 143 ADpYEPP 148 ) is shown in stick model.
- the SLP76pTYR inhibitor of the present disclosure includes residues 132- 155 of SLP76.
- FIG. 17B Electrostatic profile is shown, calculated using the APBS plugin in Pymol.
- FIG.17C Top: Organization of the domain architecture of full-length ITK showing the c-terminal Kinase domain, Src-homology 2 (SH2), the Src-homology 3 (SH3) domains, the intrinsically disordered proline-rich region (PRR), and the N- termimal Pleckstrin homology (PH) and Tec homology (TH) domains.
- FIGS. 18A-18F depict Novel peptide SLP76pTYR specifically targets SLP76:ITK signaling and enhances Treg cell development.
- FIG. 18B Total T cells stimulated in the presence of SLP76pTYR, nonspecific peptide, or vehicle alone were examined for total CD4 cells that are FoxP3 + .
- FIG.18C Quantification of three experiments as in (A)
- FIG. 18D Cell lysates were obtained from mouse T cells stimulated with anti-CD3 and anti-CD28 in the presence of SLP76pTYR, or vehicle alone.
- Lysate from stimulated cells and non-stimulated cells were examined for phosphorylated ITK, total ITK (size 50-75kDa), phosphorylated PLC ⁇ 1 total PLC ⁇ 1 (size ⁇ 155kDa), phosphorylated ERK, total ERK (size ⁇ 42kDa), phosphorylated PIC ⁇ 1, total PIC ⁇ 1, (size ⁇ 85kDa), phosphorylated AKT, and total AKT (size ⁇ 60kDa).
- n 3 and one representative experiment is shown.
- FIG.18E Cell lysates from human T cells, non-stimulated or stimulated with OKT3 for 5min in the presence of SLP76pTYR or vehicle alone, were examined for phosphorylated pPLC ⁇ 1 and total PLC ⁇ 1 on stimulated and non-stimulated T cells.
- FIG.18F Primary human T cells from PBMCs were stimulated with anti-CD3 and anti-CD28, or with PMA/Ionomycin, for 6 hours in the presence of vehicle alone or SLP76pTYR in the presence of Brefeldin A (BFA) (Webb et al., 2015). Intracellular IFN- ⁇ and TNF- ⁇ expression by CD8 + and CD4 + T cells was determined by flow cytometry. For statistical analysis we used two-way ANOVA and Student’s t test. P values are presented. See also Fig.28. [00195] More specifically, FIGS.27A-27F depict ITK inhibitors 10n and CTA056 are not specific for ITK, Related to Figure 5. (FIG.
- FIG. 27A WT mouse T cells were cultured with either 10n or vehicle, then lysed post-incubation, and lysates were western blotted for pITK (size 50-75kDa), pPLC ⁇ 1 (size ⁇ 155kDa), pERK (size ⁇ 42kDa), pAKT (size ⁇ 60kDa), and pmTOR(size 240kDa).
- FIG.27B Western blots from three experiments were quantitated and normalized to actin.
- FIG. 27C T cells from primary human PBMCs were isolated and cultured with commercially available 10n or vehicle and western blotted for pPLCg1, pERK, pAKT, and pmTOR.
- FIGS. 28A-28H depict quantitative analysis of SLP76:ITK signaling protein expression in cells treated with peptide SLP76pTYR.
- WT CD8 + and CD4 + T cells were mixed at a 1:1 ratio and transduced with a retrovirus carrying SLP76pTYR-pCherry or empty vector.
- Lethally irradiated recipient BALB/c mice were transplanted with 10X10 6 T cell-depleted BM ( TCD BM) as described, alone or together with WT CD8 + and CD4 + T cells transduced with SLP76pTYR-pCherry or empty vector- carrying virus.
- mice were also given 1x10 5 primary B-ALL- luc tumor cells as described (Cheng et al., 2016). While tumor growth was observed in TCD BM-transplanted mice that did not receive donor T cells, tumor growth was not seen in mice transplanted with either untransduced T cells, or T cells transduced with either empty viruses or SLP76pTYR-pCherry carrying viruses. Notably, mice transplanted with untransduced T cells or T cells transduced with empty virus suffered from GVHD, while mice transplanted with T cells transduced with SLP76pTYR- pCherry carrying virus survived for > 40 days post-HSCT without tumor growth, with minimal signs of GVHD (FIG.19A-E).
- FIGS. 19A-19F depict Inhibition of T cells by the peptide SLP76pTYR allows tumor clearance without inducing GVHD.
- FIG. 19A Purified WT CD8 + and CD4 + T cells were mixed (1X10 6 total) at a 1:1 ratio, and transduced with viruses containing SLP76pTYR or empty vector, then transplanted along with 1X10 5 B-ALL- luc cells and 5X10 6 T cell-depleted bone marrow cells into irradiated BALB/c mice.
- mice Host BALB/c mice were imaged using IVIS 2003 times a week. Group one received 10X10 6 T cell-depleted bone marrow alone ( TCD BM). Group two received 10X10 6 TCDBM along with 1X10 5 B-ALL-luc cells (TCDBM+B-ALL luc ). The third group was transplanted with 10X10 6 TCD BM and a 1:1 ratio of purified WT CD8 + and CD4 + T cells (1X10 6 total) along with 1X10 5 B-ALL-luc cells (TCDBM+B-ALL luc +WT CD8+CD4).
- TCDBM Group five received 10X10 6 TCDBM, a 1:1 ratio of purified WT CD8 + and CD4 + T cells (1X10 6 each) transduced with SLP76pTYR-carrying viruses, and with 1X10 5 B-ALL-luc cells (TCDBM+B- ALL luc +SLP76pTYR virus CD8+CD4)
- FIG. 19E Quantitated luciferase bioluminescence of tumor growth.
- FIG.19F Tumor incidence for each of the experimental groups. Statistical analysis for survival and clinical score was performed using log-rank test and two-way ANOVA, respectively. Note: Controls are na ⁇ ve for tumor, but transplanted with 10X10 6 T cell depleted bone marrow alone ( TCD BM) and used as a negative control for BLI. [00199] Altogether, these data demonstrate that disruption of SLP76:ITK signaling can separate GVHD from GVL. Inhibition of ITK signaling by SLP76pTYR, by specifically targeting the SLP76 and ITK interaction, allows tumor clearance and minimizes development of GVHD.
- the adapter protein SLP76 plays an essential role in regulating T cell activation downstream of the TCR by assembling a multimolecular signaling complex that includes ITK.
- the phosphorylation of SLP76 at Y145 leads to the activation and recruitment of ITK, which phosphorylates PLC ⁇ 1, leading to its activation, mobilization of calcium, and activation of the NFAT transcription factor (Sahu and August, 2009).
- T cells that carry a Y145F mutation in SLP76 fail to phosphorylate and activate PLC ⁇ 1 in response to TCR stimulation (Jordan et al., 2008) .
- T cells expressing the SLP76 Y145F mutation exhibit signaling defects downstream of TCR stimulation, not all T cell functions are lost when ITK recruitment and activation is defective.
- SLP76Y145FKI mice can clear acute LCMV infection (Smith-Garvin et al., 2010).
- the ability of T cells from SLP76Y145FKI mice to induce GVL without GVHD indicates that the SLP76:ITK pathway controls these functions.
- Several groups have reported that both na ⁇ ve CD44 lo CD4 + and CD8 + T cells can induce lethal GVHD, while CD44 hi T cells do not (Dutt et al., 2011).
- mice eventually succumbed to the symptoms of GVHD, confirming previous reports (Zhang et al., 2005).
- SLP76Y145F mice exhibit defects in the development of other T cells in addition to the IMP cells, such as iNKT (Gerth and Mattner, 2019, Muro et al., 2019) cells and gamma delta T cells (Muro et al., 2019, Navas et al., 2017), on the role of CD8 + and CD4 + T cells in GVL vs. GVHD was specifically focused on.
- both CD8 + and CD4 + SLP76Y145FKI T cells with IMP phenotype as well as those lacking the IMP phenotype exhibit GVL with reduced capacity to induce GVHD. It is likely that the altered signaling experienced by the cells with the SLP76Y145FKI mutation allows these cells to be able to have anti- tumor activity in a T cell-intrinsic manner.
- inflammatory cytokines act as mediators of acute GVHD (Lynch Kelly et al., 2015, Holler, 2002, Ju et al., 2005). Therefore, whether donor T cells with attenuated TCR signaling might reduce cytokine storm mediated by donor T cells was investigated.
- both CD8 + and CD4 + T cells from WT C57Bl/6 mice (MHC haplotype b) produced significantly higher cytokines both on a serum and a cellular level when transplanted into BALB/c mice.
- both donor CD8 + and CD4 + T cells from SLP76 Y145FKI C57Bl/6 mice produced significantly less cytokines on both a serum and a cellular level.
- Both donor CD8 + and CD4 + T cells from SLP76 Y145FKI also exhibited reduced expression of chemokine receptors compared to WT donor T cells.
- the defective migration of donor CD8 + and CD4 + SLP76Y145FKI T cells likely contributes to the attenuation of GVHD.
- the retention of T cells in secondary lymphoid organs by FTY720-mediated inhibition of S1PR1 also ameliorates GVHD while maintaining GVL effects (Villarroel et al., 2014, Liu et al., 2012).
- the chemokine receptor CXCR3 has an important role in the migration of effector T cells in GVHD model (Duffner et al., 2003).
- CX3r1, CXCr1, CCR12, CrTAM, CXCR6, CCR9, CXCR5 and CXCR4 have been shown to play a significant role in donor T cell migration to GVHD target organs (Barrett, 2015, Castor et al., 2012, Hsiao et al., 2020).
- CXCR6 deficiency or blockade of the CXCR3 and CXCR6 ligands attenuates GVHD (Duffner et al., 2003), and importantly, the GVL effect is still maintained under these conditions (Sato et al., 2005).
- blocking T cell migration by chemokine receptor blockade could be beneficial in the treatment of GVHD after allo-HSCT. It is shown that specific targeting of the SLP76: ITK interactions can be achieved to potentially differentially modulate GVL and GVHD by pharmacologic agents.
- SLP76pTYR inhibition of ITK signaling also enhances Tregs frequency in vitro, confirming the peptide’s ability to affect ITK signaling and T cell effector functions.
- This SLP76pTYR peptide significantly reduced IFN- ⁇ and TNF- ⁇ production by TCR stimulated primary human T cells isolated from PBMCs.
- Treatment of murine donor T cells with SLP76pTYR prior to transfer resulted in tumor clearance without inducing GVHD.
- Future therapies involving our novel SLP76pTYR peptide inhibitor and small molecule inhibitors could potentially be an effective strategy for enhancing GVL while avoiding GVHD.
- more selective ITK inhibition using our SLP76pTYR peptide could be beneficial in the treatment of autoimmune diseases while maintaining T cell effector functions.
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